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
Caenorhabditis elegans
Drosophila melanogaster
Californias governorus

5 Key Signal transduction pathways
Are 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 (me)
TGF-ß (you)
Hedgehog (you)
Notch (you)

Signal transduction pathways
RTK=
Receptor tyrosine kinases
Src=non-receptor tyrosine kinase
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?
Ras

Step by step--think dominos
Ras
Ras

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
Kurt Cobain
A new graduate from Harvard

Model organisms teach us about ourselves
Mus musculus
Xenopus laevis
Caenorhabditis elegans
Drosophila melanogaster
Californias governorus

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 all the cells needed to see the world
Including the eight photoreceptors

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 sev ts sev ts sev ts
; enh
22.7
o C 24.3
o C 22.7
o 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 sev ts sev ts sev ts
; enh
22.7
o C 24.3
o C 22.7
o 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 sev ts sev ts sev ts
; enh
22.7
o C 24.3
o C 22.7
o C
*/+
Gian Garriga

The mutations identified were candidates to encode things in the RTK pathway

Wow--Ras, the adapter Grb2 and a GEF are all 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
Physiology and
Medicine 2002
Bob Horvitz John Sulston Sydney Brenner lineage and programmed cell death
Chemistry 2008
GFP
Marty Chalfie
Physiology and
Medicine 2006
RNAi
Andy Fire Craig Mello

A simple model for organogenesis
Formation of the vulva in C. elegans
( “ ask Gidi Shemer what he did in graduate school?
” ) early oocytes embryos vulva sperm
Only 22 cells!
oocytes

The Key Players
One gonadal anchor cell ( AC )
6 vulval precursor cells ( VPCs )
The anchor cell induces vulva l 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

No vulva induction
Mutants with no vulval signaling vulvaless (Vul)
Mutants with too much vulval signaling multivulvae (Muv) wild type

Loss of function mutants in the signaling pathway vulvaless (Vul)
Gain of function mutants in the signaling pathway or loss-of-function mutations in pathway negative regulators multivulvae (Muv)

A Screen for Vul and Muv mutants
Bob Horvitz
Nobel Prize physiology and medicine 2002 some examples
Vul let-23 lin-3
Muv lin-15 let-60
Next step: cloning and sequencing the genes

The first two vul mutations identify the anchor cell signal and its receptor
let-23 is an EGFR homologue = RTK
lin-3 is an EGF homologue

Lin-3 is expressed in the AC
Let-23 is expressed in all the VPCs
Alberts et al. 21-44
Where do they function?

Muv lin-15 let-60 lin-15 lin-15 was found to be an negative regulator of vulval induction ( loss of function mutation)

let-60 was found to be a gain of function mutation that promoted vulval induction
Next step: clone the let-60 gene

First evidence: Ras has an in vivo role as part of the RTK pathway

The ras gain-of-function mutation =
Glycine 13 Glutamine
Sound familiar?

The ras gain-of-function mutation =
Glycine 13 Glutamine
Sound familiar?

The gain-of-function mutation =
Glycine 13 Glutamine
Constitutively active
Ras
G13Q
All the VPCs make vulvae

How can we find the rest of the proteins in the pathway? lin-15
?
Suppressor and enhancer screens
[suppressors (or enhancers) of the mutated phenotype]

For example, second mutations that turn
Muv mutants into normal or Vul worms
Vul = “ bag of worms ”
Normal vulva ”
Muv

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)
Approved for treatment of advanced renal cell carcinoma (Jan. 2006) and approved for inoperable hepatocellular carcinoma (Nov. 2007)

The RTK-Ras pathway offers drug targets for cancer treatment e.g., or the Raf kinase inhibitor Vemurafenib
Approved for treatment of
Late stage melanoma August 2011) and approved for inoperable hepatocellular carcinoma (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