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 ourselves
Mus musculus
Xenopus laevis
Californias governorus
Caenorhabditis elegans
Drosophila melanogaster
5 Key Signal transduction pathways
Critical for development and homeostasis
All are involved in Cancer
5 Key Signal transduction pathways
Critical for development and homeostasis
All are involved in Cancer
RTK- Ras (me)
Wnt (you)
TGF-ß (you)
Hedgehog (you)
Notch (not this time)
Signal transduction pathways
RTK
Receptor tyrosine kinases
Src
RTKs
In the absence of Ligand
RTKs are monomers
with an inactive kinase
Ligand binding activates RTKs
by dimerization
Lodish et al. Fig. 20-21
RTKs are their own substrates-i.e., they autophosphorylate
Remember SH2 domains?
What did they bind??
SH2 domains allow “effector” proteins
to bind activated receptors
One adaptor with an SH2 domains is Grb2
It also has SH3 domains--what do they bind?
One adaptor with an SH2 domains is Grb2
It also has SH3 domains--what do they bind?
Step by step--think dominos
Remember-Ras is anchored to the membrane
through a lipid
And SOS is a GEF--remember them??
Each activated protein activates the next
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
Each activated protein activates the next
MAPK/ERK enters nucleus
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
Each activated protein activates the next
MAPK/ERK enters nucleus
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
Each activated protein activates the next
MAPK/ERK enters nucleus
Figure 15-60 Molecular Biology of the Cell (© Garland Science 2008)
The RTK pathway
We made it to the 90s
10 years-old
Britney Spears
Travolta is still dancing
A new graduate
from Harvard
Kurt Cobain
Model organisms teach us about ourselves
Mus musculus
Xenopus laevis
Californias governorus
Caenorhabditis elegans
Drosophila melanogaster
The eye of a fly:
One Key to
learning how
Ras and RTKs work
Lodish et al. Fig. 20-24
Did you say flies?
Did you
say flies?
This is all
I need to know
But wait--flies
Helped us
Understand
The single
Most important
Human
oncogene
Lodish et al. Fig. 20-24
I told you the RTK pathway is key
in MANY developmental decisions
Each ommatidium contains the cells
needed to see the world
These cells choose fate one by one,
each telling the next what fate to adopt
sevenless mutants lack an R7
photoreceptor
Wild-type
sevenless mutant
Sevenless encodes an RTK
Lodish et al. Fig. 20-25
To get a cellular response,
There is a threshold level of pathway activity
R7 present
sev threshhold
R7 absent
wild type sev-
sevts
sevts
22.7o C 24.3o C
sevts; enh*/+
22.7o C
Gian Garriga
Scientists figured out
how to tune RTK activity
using a temperature sensitive mutant
R7 present
sev threshhold
R7 absent
wild type sev-
sevts
sevts
22.7o C 24.3o C
sevts; enh*/+
22.7o C
Gian Garriga
They then looked for mutants
In other genes that would drop
Pathway activity below the threshold
R7 present
sev threshhold
R7 absent
wild type sev-
sevts
sevts
22.7o C 24.3o C
sevts; enh*/+
22.7o C
Gian Garriga
The mutations identified were candidates
To encode things in the RTK pathway
Wow--Ras, an adapter and a GEF
are in the RTK pathway!
Sos
Grb2
Ras
Further, Ras acts downstream of
the RTK Sevenless
Lodish et al. Fig. 20-25
The signal transduction pathway
should look familiar
Grb2
Alberts et al. Fig. 15-53
Because that’s how we
figured it out!
Grb2
Alberts et al. Fig. 15-53
Flies were bad enough,
but worms!?
Caenorhabditis elegans
Luckily the Nobel Committee
thinks they are cool
Caenorhabditis elegans
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
(“ask Gidi what he did in graduate school?”)
early oocytes
embryos
sperm
vulva
Only 22 cells!
oocytes
The Key Players
One gonadal anchor cell (AC)
6 vulval precursor cells (VPCs)
The anchor cell induces vulval fates
Sherwood and Sternberg (2003) Dev Cell
Cell Induction
The AC signals the VPCs to adopt vulval fates
Only 3 VPCs will actually form the vulva
How can we figure out
how to build a vulva?
GENETICS!
wild type
No vulva induction
Mutants with no
vulval signaling
vulvaless (Vul)
Mutants with too much
vulval signaling
multivulvae (Muv)
Loss of function mutants
in the signaling pathway
vulvaless (Vul)
Gain of function mutants
in the signaling pathway
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 and sequencing the genes
The first two vul mutations
identify
the anchor cell signal
and its receptor
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
How can we find the rest
of the proteins in the pathway?
lin-15
Other mutations?of ras
?
Suppressor and enhancer screens
[suppressors (or enhancers) of the mutated phenotype]
For example, second mutations that turn
Muv mutants into normal Vul worms
Vul = “bag of worms”
Normal vulva”
Muv
This worked GREAT!
lin-15
This worked GREAT!
lin-15
The fly and worm work allowed cell biologists and biochemists
to return to mammalian cells
to identify the ways these new proteins worked as machines
The RTK-Ras pathway also offers
drug targets for cancer treatment
Alberts et al. Fig. 21-44
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)
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)
Increased survival in Phase III liver cancer trial
and approved for inoperable form (Nov. 2007)
Summary
- Cellular oncogenes = viral oncogenes
Summary
- Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small
GTPase, acting as a molecular switch
Summary
- Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small
GTPase, acting as a molecular switch
- Ras is a major component of the RTK pathway
Summary
- Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small
GTPase, acting as a molecular switch
- Ras is a major component of the RTK pathway
- Basic and Clinical Science provide
a VERY powerful partnership