Cellular Oncogenes
The Story of Ras
Last week, we made it to the 70s
Viral Oncogenes Pave the Way
c-src
proto-oncogene
and h-ras, v-myc , etc.
v-src
oncogene
At the time, no known viral tumors in humans.
Carcinogenes can cause cancer.
Maybe oncogenes can rise independently of viruses.
Bob Weinberg
infect with DNA from a cell?
Viral DNA can be transfected into cells using
Calcium Phosphate
Adenovirus
Viral DNA can be transfected into cells and
induce tumors
Late 50s
Late 70s
Chiaho Shih
Bob Weinberg
Figure 4.2 (part 2 of 2) The Biology of Cancer (© Garland Science 2007)
DNA from chemically treated Mouse
Cells can Transform Normal Mouse Cells
foci are formed and cells in foci show tumor characteristics
Without a Virus: Cells can be Transformed
It’s time to move on- the 80s
Could This Work for Human Tumors?
transfection of DNA from human cancer cell lines
Human Cancer
Cell Line
Chiaho Shih
Figure 4.8 The Biology of Cancer (© Garland Science 2007)
Bob Weinberg
DNA from Human Cancer Cell Lines
can Transform Mouse Cells
a focus after transfection
cells from the focus
cells near the focus
Next Challenge: How to Isolate the Human DNA?
Figure 4.3 The Biology of Cancer (© Garland Science 2007)
How to Find a Needle in a Haystack
mouse sequence
Figure 4.8 The Biology of Cancer (© Garland Science 2007)
How to Find a
Needle in a
Haystack
bacteriophage genomic library
Lodish et al. Fig. 24-4
The Race is ON
Wigler lab
December 1981
Barbacid lab
Weinberg lab
An Oncogene is Cloned From a Human Tumor !!
Independent of any viral infection
the cloned gene is ~25,000 bp
no PCR available
cutting, ligating, sequencing
what about a negative control ?
In the mean time, why not verify that the cellular
oncogene is not a viral oncogene?
Why to expect a negative answer?
14 v-oncogenes, ~50,000 human genes
no evidence for a human tumor virus
we don’t want the results to be positive
Bob Weinberg
Luis Parada
Southern Blot Analysis
what probe should we use ?
v-src, v-ras (h-ras), v-myc, etc.
Figure 4.4 The Biology of Cancer (© Garland Science 2007)
The Transforming Oncogene is Ras
human RAS
mouse RAS
Der et al. PNAS 82
Channing Der, UNC
Ras Acts As a Molecular Switch
Lodish et al. Fig. 20-5
How is oncogenic ras different from
the normal proto-oncogene?
A new race starts
Figure 4.9 The Biology of Cancer (© Garland Science 2007)
Table 4.2 The Biology of Cancer (© Garland Science 2007)
G12V Mutation Prevents Endogenous and
GAP-Stimulated GTPase Activity
The Ras Oncogene is constitutively Active
G12V Mutation Prevents Endogenous and
GAP-Stimulated GTPase Activity
The Ras Oncogene is constitutively Active
G12V
Lodish et al. Fig. 20-5
Why would constitutively active Ras
lead to Cancer ?
How does Ras act in our body, in vivo ?
From Cell Culture to Model Organisms
Model Organisms Teach Us About Us
Mus musculus
Xenopus laevis
Californias governorus
Caenorhabditis elegans
Drosophila melanogaster
Signal Transduction Pathways
Important for development and homeostasis.
All involved in Cancer.
RTK
Wnt
TGF-β
Hedgehog
Notch
Signal Transduction Pathways
RTK
Receptor Tyrosine Kinases
Src
RTKs
Ras is anchored to the membrane by a lipid modification.
MAPK/ERK enters nucleus
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
The RTK Pathway
How Do We Know All of This?
Model Organisms and Genetics
The Model: The Compound Eye of the Fly
The compound eye of insects is composed of 750
individual units: ommatidia.
Each ommatidium is composed of 33
cells, including pigment cells, cone
cells and in the middle:
8 photoreceptor neurons (the rods
and cones of flies).
The R8 cell differentiates first.
The R7 cell differentiates last.
The Sequential Induction Model
differentiation is established through cell-cell
interactions
Figure 15-57 Molecular Biology of the Cell (© Garland Science 2008)
How Do We find The Molecules Involved?
Mutants
Genetic Approach
Reverse genetics
Knockdown a specific
candidate gene and test the
consequences.
Forward genetics
Mutagenize animals
randomly and select for
mutations that affect eye
differentiation.
Genetic Screens
treat flies with a mutagen (e.g. EMS)
screen for mutants in eye differentiation
assays
rough vs. smooth eyes
behavioral reaction to
different wave lengths
Genetic Screens
mutants
cloning
sequence
comparison
suggested function
antibodies and GFP constructs
expression and localization
sevenless (sev) encodes a TM protein with a cytoplasmic
kinase domain (RTK?)
bride of sevenless (boss) encodes a TM protein (ligand?)
Mosaics analysis shows the molecular interactions between
Boss in R8 and Sev in R7.
No identified downstream genes to Sev and
Boss in these screens.
Why?
Sev
a downstream gene
eye differentiation
viability
-/-
Other developmental events
How can we find mutants in the Sevenless
pathway w/o affecting viability?
Michael Simon and Gerald Rubin, Cell 1991
We made it to the 90s
10 years-old
Britney Spears
Travolta is still dancing
A new graduate
from Harvard
Kurt Cobain
Boss
Sev
protein X
phosphorylated
protein X
Partially active
Sev
50% of
Protein X
How can we find mutants in the Sevenless
pathway w/o affecting viability?
In a sensitized genetic background, where the pathway is
only partially active,
reducing another protein’s concentration by 50% might
have an effect on eye development.
We will look for dominant enhancers mutants that are
hetrozygotes : m/+
Looking for other players in the pathway
using genetics.
Dominant Enhancer Screen
R7 present
Sev threshhold
R7 absent
wild type
sev-
sevts
22.7o C
Lecture slide of Gian Garriga, UC Berkeley
sevts
24.3o C
sevts; enh*/+
22.7o C
The Final Ingredient
Where do we find ts sev mutants?
induced conserved mutations from src
mutants in the sev gene:
formed sev ts mutants
Michael Simon and Gerald Rubin, Cell 1991
Looking for other players in the pathway
using genetics.
Dominant Enhancer Screen
R7 present
Sev threshhold
R7 absent
wild type
sev-
sevts
22.7o C
Lecture slide of Gian Garriga, UC Berkeley
sevts
24.3o C
sevts; enh*/+
22.7o C
7 genes were found that might function in the
RTK pathway, including:
1) Son Of Sevenless (Sos)
Sos sequence suggested it is a Ras GEF
2) Ras !!!
Looking for other players in the pathway
using genetics.
?
R7 present
Sev threshhold
R7 absent
wild type
sev-
sevts
22.7o C
sevts
24.3o C
sevts; m*/+
22.7
24.3oo CC
dominant suppressors
genes involved in reducing RTK pathway activity
Raf
rapidly accelerated fibrosarcoma
First, isolated as a viral oncogene.
Sev
a downstream gene
eye differentiation
viability
-/-
Other developmental events
Figure 1
WT
(slightly rough)
raf(-)
(dominant rough)
sev(-); ectopic
raf
Sev
Ras
Raf
normal
eye
sev(-);
sev(-); ectopic
ectopic ras
raf
(dominant rough) (dominant rough)
sev(-); ectopic (suppresses
ras; raf(-) dominant rough)
Figure 1
(R7+)
Sev
WT
(no R7)
sev (-)
(no R7)
raf(-)
(no R7)
sev(-); normal
raf
Ras
Raf
Sina
normal
eye
sev(-); ectopic sev(-); ectopic
raf
raf; sina(-)
(R7 ++)
(no R7)
sev(-); ectopic
sev(-);
ras; raf(-)
ectopic ras
(R7 ++)
no R7
Figure 2
western blot, verifying the presence of
the proteins
Ectopic Raf has increased kinase activity.
Figure 3
early
late
sev(-)
no R7
WT
R7 (+)
sev(-);
ectopic Raf
R7 (+) and
even early
Raf is necessary and sufficient for R7 differentiation
Figure 4
Flies were bad enough,
but worms ??
Caenorhabditis elegans
exploratorium
Nobel Prize
lineage and
programmed
cell death
Physiology and
Medicine 2002
Bob Horvitz John Sulston Sydney Brenner
Chemistry 2008
Physiology and
Medicine 2006
GFP
RNAi
Marty Chalfie
Andy Fire
Craig Mello
A simple model for organogenesis.
Formation of the Vulva in C. elegans
early oocytes
embryos
sperm
vulva
Only 22 cells!
oocytes
early oocytes
embryos
sperm
vulva
oocytes
The Key Players
one gonadal anchor cells (AC)
6 vulval precursor cells (VPCs)
The anchor cell induces vulval fates.
Sherwood and Sternberg (2003) Dev Cell
How do we know this?
killing the AC with a laser
no vulva is formed
vulvaless worm
(bag of worms)
Cell Induction
The gonadal anchor cell (AC) signals vulval precursor
cells (VPCs) to adopt vulval fates
3 VPCs will form the vulva, while the other 3 will
fuse to the epidermis
So, why call all of them VPCs ?
All the VPCs are competent to form a vulva
vulva
A ligand gradient is secreted from the AC.
The receptor for the secreted ligand is expressed in all the VPCs.
Vulva Formation in C. elegans
cell migration
cell fusion
ring formation
How can we learn about the molecular
mechanisms involved in vulva formation?
Mutants
wild type
No vulva induction
mutants with no
vulval signaling
vulvaless (Vul)
mutants with too much
vulval signaling
multivulvae (Muv)
A Screen for Vul and Muv Mutants
some examples
Bob Horvitz
Nobel Prize
physiology and
medicine 2002
Vul
Muv
let-23
lin-15
lin-3
let-60
Next step: cloning of the genes
Vul
let-23
let-23 is an EGFR homologue = RTK.
lin-3
lin-3 is an EGF homologue.
Where do they function?
Lin-3 is expressed in the
AC.
Let-23 is expressed in all
the VPCs.
Alberts et al. 21-44
Muv
lin-15
lin-15
let-60
lin-15 was found to be an inhibitor of vulval induction
(loss of function mutation)
let-60 was found to promote vulval induction (gain of
function mutation)
Next step: clone the let-60 gene
First evidence: Ras has an in vivo role as part of
the RTK pathway.
The gain-of-function mutation = Glycine
13 Glutamine
Constitutively active
Ras
All the VPCs make vulvae.
G13Q
let-23
let = lethal
Their good fortune: the screen yielded a weak mutant
allele of let-23, affecting vulva formation, but not
viability
This suggests that other genes might be there, but those
are essential for viability
How can we find such genes?
lin-15
Other mutations?of ras
Suppressor Screens
suppressors of lin-15 Muv mutants
?
lin-15
suppressor of lin-15 Muv mutants
Let-23
Let-60
GRB-2
Raf
others
Model Organisms Teach Us About Us
Mus musculus
Xenopus laevis
Californias governorus
Caenorhabditis elegans
Drosophila melanogaster
The RTK Pathways is Highly Conserved
The RTK-Ras pathway offers
drug targets for cancer treatment
e.g., the Raf kinase inhibitor sorafenib
(also inhibits the RTKs VEGFR, PDGFR, and Kit)
In clinical trials for melanoma and lung cancer
Approved for treatment of
advanced renal cell carcinoma (Jan. 2006)
Alberts et al. Fig. 21-44
Increased survival in Phase III liver cancer trial
and approved for inoperable form (Nov. 2007)