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BIOL 445 Cancer Biology
Spring 2015
Mark Peifer and Bob Duronio
BIOL 445
In Bio 445 we combine
the approaches of Bio 202 + Bio 205
with the current scientific literature
to study the family of diseases
known as cancer
You may not believe it but by the end of the semester
This will make sense!
Hanahan and Weinberg, Cell 100:57-70 (2000)
Virtually everything you’ll need is found at:
http://www.bio.unc.edu/Courses/2015Spring/Biol445/
It’s a REALLY good idea to
look through this carefully THIS WEEK
AND
Check back frequently for updates or changes
BIOL 445
Textbooks
-The Biology of Cancer by Robert Weinberg 2nd Edition
What are you responsible for?
- Lectures (attendance AND participation)
- Papers and assigned textbook reading(on website)
-Your project- literature search and presentation
Grading
- Exams (20% X 2 midterms + 25% final) = 75
- Presentations = 15
- Classroom discussion & in-class evaluations = 10
Your Project
- Choose a topic
- Read the posted review paper
- Do a thorough literature research, including
primary data on the gene and the disease
- Make a poster and present it to your peers
- The final exam will cover ALL posters
As you learned in the Online Introduction
and the assigned readings for today,
Cancer is a family of diseases
caused by our own cells gone wrong
Cancer is number 2
and rising relative to heart disease!
CDC
But as we’ll see, Cancer is
not one disease, its many
ONS=Other nervous system.
Source: American Cancer Society, 2009.
In the Online Introduction
You learned about
- Properties of cancer cells
- Tumor progression
- What causes cancer?
- Accumulation of mutations
- Molecular genetics of cancer
Cancer often starts with a
single mutation
However
One mutation is not enough !!
Heard of
natural selection?
What types of genes are mutated in cancers?
What types of genes are mutated in cancers?
Two broad categories
Oncogenes
Mutational activation of proteins
that normally Promote cell proliferation
What types of genes are mutated in cancers?
Two broad categories
Oncogenes
Mutational activation of proteins
that normally Promote cell proliferation
Tumor suppressor genes
Mutational inactivation of proteins
that normally inhibit cell proliferation
Oncogenes
proto-oncogene
oncogene
A proto-oncogene: a normal cellular gene that
can become an oncogene,
upon mutational change
As you know from Bio 205,
cells instruct one another
via cell-cell signals
Be
Posterior!
Turn on
new genes!
Signal transduction
moves information
from the cell surface
to the nucleus
& other cellular targets
Cell surface
Yes
Ma'am!
Nucleus
Turn on
new genes;
pass it on
Turn on
new genes;
pass it on Turn on
new genes;
pass it on
Cell surface
Signal transduction
occurs in a
series of steps
Turn on
new genes;
pass it on
Yes
Ma'am!
Nucleus
Proliferate!
Proliferate!
Cell surface
Cell-cell signals
can regulate
cell proliferation
Proliferate!
Proliferate!
Yes
Ma'am!
Nucleus
Cell-cell signals
can regulate
cell proliferation
Cancer at the Cellular
Level
Signal transduction drives
information from the cell
surface to the nucleus
& other cellular targets
It’s time for
a Bio 202 review
How many copies
of each gene
do we have?
Are most mutations
dominant or recessive?
Why?
Oncogenes result from rare dominant mutations
That lock signaling machinery in the ON state
Genes did not evolve
to cause cancer!
Src in its normal context
Normal skin cell tightly adherent to ECM
Alberts et al.
Signaling is OFF
Wounding->platelet recruitment->
cell migration and proliferation
Alberts
et al.
Signaling is ON
Oncogenes
Cellular signaling machinery is stuck ON
One mutated copy =
over active protein =
over proliferation, oversurvival, etc.
What types of genes are mutated in cancers?
Two broad categories
Oncogenes
Tumor suppressor genes
Mutational inactivation of proteins
that normally inhibit cell proliferation
Oncogenes
The good guys become bad guys
Tumor suppressor genes
We lose the good guys
Rb puts the brakes on
cell proliferation by acting as
A transcriptional co-repressor
Rb
E2F DP
DO NOT
transcribe genes
required for DNA
replication
Rb phosphorylation
frees E2F/DP to turn on genes
required for proliferation
P
P
E2F DP
Transcribe gene
required for DNA
replication
Retinal tumor in patient that
inherited a mutant copy of Rb
Lodish et al. Fig. 24-11
Retinoblastoma is inherited in
a dominant fashion
Lodish et al. Fig. 24-12
However, at a cellular level
Retinoblastoma is Recessive!
Normal individual with
two functional copies of Rb gene
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
Rb
In rare cell one copy mutated,
but second copy remains
The remaining Rb
still puts the brakes on
cell proliferation
Rb
DP
E2F
DO NOT
transcribe genes
required for DNA
replication
It’s only when both copies of Rb are knocked out
in a single cell that a tumor develops
In person with hereditary
Retinoblastoma, all cells start
with one mutant copy
Rb
Rb
Rb
Rb
If somatic mutation occurs, cell
has no functional Rb
and tumor develops
Loss of functional Rb
frees E2F/DP to turn on genes
required for proliferation continuously
E2F DP
Transcribe gene
required for DNA
replication
Thus oncogenes and tumor suppressors
Differ in both cellular function and genetics
dominant
recessive
Figure 20-27 Molecular Biology of the Cell (© Garland Science 2008)
The story of Src
What Viruses and Nobel Laureates
Taught Us About Cancer
No thank you!
Peyton Rous
discovered
a virus that causes
cancer in chickens
The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor
The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor
Nobel Prize in
Physiology or
Medicine 1966
The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor
But what’s a virus???
Carcinogens
Chemicals can directly induce cancer
1920s
Viral Infection Out
Yamagiwa
Chemical Induction
In
30 Years Later: Rebirth of RSV research
RSV can transform cells in culture
RSV stock
Howard Temin
Harry Rubin
Immortality
Studying cancer at the cellular level
RSV infection
Changed cells
No contact inhibition
on cell division
No contact inhibition of cell division
Normal
Normal
RSV
infected
= Cancer
RSV
infected
= Cancer
But how???
Normal
Normal
RSV
infected
= Cancer
RSV
infected
= Cancer
I HOPE you remember the central dogma
This is one of those biology facts
That you need to have permanently stored
The central dogma
DNA
Transcription
mRNA
Translation
Protein
RSV is a retrovirus
These viruses reverse the central dogma,
making a DNA copy of their RNA genome
and inserting it into your DNA
Alberts et al. Fig. 24-23
Nobel Prize in
Physiology and
Medicine 1975
Howard Temin and David Baltimore
Alberts et al. Fig. 24-23
Your genome is a retrovirus graveyard:
living and dead retroviruses make up
8%of your genome,
with ~100,000 whole or partial copies!
Alberts et al. Fig. 24-23
NEXT Breakthrough discovery
Retroviruses can cause cancer by picking up
mutated versions of normal cellular genes
Alberts et al. Fig. 24-23
The paper
that created
two more
Nobel laureates
and founded the
modern field of
Cancer biology
Let’s take a very short detour
Retroviruses can also cause cancer by
inserting next to and thus
activating the expression of proto-oncoge
Retroviral insertion
sites in different tumors
Transcribe to mRNA
5 kilobases
exons
wnt-1 gene
Alberts et al. Fig. 22-24
Two mechanisms of gene activation
by retroviral insertion
Lodish et al. Fig. 24-10
OK—Back to src
You know mis-expressing this gene can
Initiate cancer
What do you want to know now??
So, what job does
the protein encoded by src
do within the cell?
The first BIG step:
using antibodies to
immunoprecipitate the
v-Src protein
This led to the discovery that Src
is post-translationally modified
This led to the discovery that Src
is post-translationally modified
What’s translation??
Protein kinases and protein phosphatas
add and remove phosphate groups from
target proteins
Lodish et al. Fig. 20-5
Adding labeled ATP to a precipitated Src showed
that Src can phophorylate a substrate
Src is a kinase
in the presence of
P32-ATP
A substrate is
phosphorylated
Which amino acids can be phosphorylated
And Why those amino acids??
Src is a Tyrosine Kinase
As a kinase, it can affect many cellular events
Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008)
Normally, Src kinase intrinsic activity is low
What makes Src so active in transformed cells?
Western Blot with antibody
that recognizes
Tyr phosphorylated
proteins
The structures of c-src and v-src
provided an important clue!
Lodish et al. Fig. 24-17
Src contains three domains
that are shared with other proteins
Binds polyproline motifs
Phosphorylates
other proteins
Binds peptides phosphorylated on Tyr
Scientists have determined
the precise 3-dimensional structure of S
Xu et al. Nature. 1997 385:595-602
Tyrosine phosphorylation of the C-terminus
creates an intramolecular
and inhibitory interaction
Lodish et al. Fig. 24-17
Src is normally
inactive due to
intramolecular
inhibition
Lodish et al. Fig. 24-17
Recent work has provided
a more detailed model of Src activation
Closed = OFF
Open = ON
Cowen-Jacob et al. Structure 13, 861-871 (200
v-src lacks the C-terminal Tyr
and thus cannot be inactivated!
Lodish et al. Fig. 24-17
Activation of Src
has multiple consequences
From Schwartzenberg, Oncogene 17, 1463-1468 (1
Where is Src within cells?
This is a covalently attached lipid
what might that mean?
Myristylation of Src is
essential for transformation
Recent work has provided
a more detailed model of Src activation
Cowen-Jacob et al. Structure 13, 861-871 (200
c- Src is a tyrosine kinase
What does it do in the cell?
What are its targets?
Remember, we are still in the late 70s
Bishop and Varmus
Identifying The Targets of Src-look for
Proteins ONLY modified by biologically active Src
Western blotting with antiphosphotyrosine antibodies
V = v-Src transfected cells
2A/V = non-myristylated v-Src
transfected cells
Reynolds et al. MCB (1989)
Identifying The Targets of Src-look for
Proteins ONLY modified by biologically active Src
Western blotting with antiphosphotyrosine antibodies
V = v-Src transfected cells
2A/V = non-myristylated v-Src
transfected cells
p120 catenin: modulates cellcell adhesion
Reynolds et al. MCB (1989)
Identifying the targets of Src
- p120 catenin: modulates cell-cell adhesion
- Cortactin A: regulates actin polymerization
- Focal Adhesion Kinase: involved in cell-matrix
interactions
Mike Schaller, ex-UNC
Src modulates both cell-cell
and cell matrix adhesion: The basics
Cell-cell
junctions
Cell-matrix junctions Basal lamina
Src modulates both cell-cell
and cell matrix adhesion: The basics
Lodish et al. Fig. 22-2
Epithelial cells secrete a special ECM
called the basal lamina
Epithelial cells
Basal Lamina
Alberts et al. Fig. 19-54
Cells interact with the ECM
via Focal adhesions, which also ancho
the actin cytoskeleton
Focal
Adhesions
(orange)
Actin: Green
Alberts et al. Fig. 17-42
Focal adhesions are
linked to the
actin cytoskeleton
Alberts et al.
Fig. 16-75
A complex network
of proteins links
the focal adhesion to act
and regulates
actin polymerization
Alberts et al.
Fig. 16-75
Focal adhesions are sites of
intense protein tyrosine phosphorylatio
Focal
adhesions
Actin: Green
Phosphotyrosine: Red
An oversimplified model of Src function
Normal skin cell tightly adherent to ECM
Wounding->platelet recruitment->
cell migration and proliferation
Alberts et al.
A less oversimplified model
Migratory growth factors
Extracellular matrix
e.g., EGF, PDGF
RTKs
Src
Integrins
FAK
PI-3kinase
Adaptors
Actin
Remodel cell-matrix
junctions -> cell motility
From Jones et al. Eur J. Cancer
36, 1595-1606 (2000)
FAK is recruited by integrins to the
membrane and is autophosphorylated
- Src binds to
phosphorylated FAK
- Src changes conformation
and becomes active
- Src further phosphorylates FAK
- Src-FAK phosphorylate target proteins
Src and FAK act together to regulate other focal
adhesion proteins
Src-FAK signals to regulate adhesion turnover
Src-FAK active = less adhesion, more migration
If Src is a critical regulator
of cell adhesion, what happens to
an animal without any Src?
Cell 1991 64:693-702
Targeted disruption of
the c-src proto-oncogene
leads to osteopetrosis in mice.
Soriano P, Montgomery C,
Geske R, Bradley A.
Why is this phenotype so modest?
Redundancy!!
Src has two very close relatives:
Fyn and Yes
Different
Src family kinases
work downstream of
different receptors
Alberts et al. Fig. 23-54
Fyn mutant mice are viable but
have defects in
myelination of brain neurons
Yes mutant mice are viable but
with subtle changes in B-cell function
Src; Fyn; Yes triple mutant mice die
at embryonic day 9.5 with multiple defects
Triple mutant
Wild-type
However, triple mutant cells
still make focal adhesions
However src; fyn; yes (SYF) triple mutant cells
fail to migrate!
Scratch assay
Scientists have determined
the precise 3-dimensional structure of Src
Active
site
This aided identification of kinase inhibitors
that block Src action
Active
site
SU6656
In leukemia, adding Src inhibition
to inhibition of the related kinase Abl
improved prognosis in phase II trials and was FDA
help get around drug resistance in CML
dasatinib
Ottmann et al. Blood 110, 2309 (2007)
This same Src inhibitor is in
Phase II trials for advanced breast cancer,
melanoma and advanced sarcomas
dasatinib
Ottmann et al. Blood 110, 2309 (2007)
Another Src inhibitor is in Phase I/II trials for
metastatic pancreatic, breast,
ovarian, and prostate cancers
Active
site
AZD0530