Cellular Oncogenes
We made it to the 70s
Viral oncogenes pave the way
c-src
proto-oncogene
v-src
oncogene
Viral oncogenes pave the way
c-src
proto-oncogene
v-src
oncogene
The concept:
-Viruses kidnap a normal proto-oncogene
-During the “kidnapping”, the mutated protopncogene became an oncogene
-A new viral infection inserted an oncogene
into the recipient, leading to cancer
However….
At the time, there were
no known viral tumors in humans
We ultimately want to know the cause of
human diseases like colon cancer
rather than curing chicken sarcomas
http://www.clevelandclinic.org/registries/inherited/fap.htm
One oncogene mutated in the progression
to malignancy is K-ras
Lodish
et al.
Fig. 24-6
Onto the stage stepped a bright and ambitious
young assistant professor with a crazy idea
Bob Weinberg
But how can we find oncogenes that are
not viral oncogenes?
But how can we find oncogenes that are
not viral oncogenes?
Add DNA from
carcinogen-treated cell?
First you need a way
to get DNA into cells
How would you do that?
DNA can be “transfected” into cells using
Calcium Phosphate
As you know, Oncogenes relieve contact inhibition.
This can be visualized in culture
by “focus formation”
Transfect with oncogene
J Biol Chem 2002 277:10813-23 Fiordalisi et al.
J Virol 2000 74:1008-13 Yoshioka et al.
One can then test if the cells induce tumors
But it’s a crazy idea
1. you need to find
the right needle in
a very large haystack
2.What if you need to find
two or more needles??
Chiaho Shih
Bob Weinberg
Maybe it wasn’t
So crazy after all!
Soon everybody
was trying it!
Chiaho Shih
Bob Weinberg
DNA from chemically treated mouse cells
can transform normal mouse cells
a focus after transfection
cells from the focus
cells near the focus
Without a virus: cells can be transformed
It’s time to move on- the 80s
Could this work for human tumors?
Transfect DNA from human cancer cell lines
Human Cancer
Cell Line
Chiaho Shih
Bob Weinberg
Human Cancer
Cell Line
Somewhere here, among the normal mouse
genes, we have a human oncogene
Low transformation rates suggest that we are
dealing with one, single oncogene
How to find a needle in a haystack
Figure 4.8 The Biology of Cancer (© Garland Science 2007)
How to find a needle
in a haystack?
Generating a bacteriophage
genomic library
Searching for the one
human gene among many
mouse genes
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 !!
But what does it encode?
The cloned gene is ~25,000 bp
no PCR available
No way to sequence 25 kilobases
Let’s try a long-shot shortcut--what if the cellular
oncogene is one of the known viral oncogenes?
Its very unlikely
- 14 v-oncogenes, ~30,000 human genes
Bob Weinberg
Luis Parada
Southern blot analysis
What probe should we use ?
human cells
mouse cells
Just go through the viral oncogenes
one by one
human RAS
mouse RAS
Der et al. PNAS 82
Channing Der, now at UNC
human cells
mouse cells
The transforming oncogene is Ras
Cellular oncogenes = Viral oncogenes
Mouse cells
transformed by a human oncogene
human RAS
mouse RAS
Der et al. PNAS 82
Channing Der, UNC
c-ras
v-ras
(viral)
(cellular )
Proto-oncogene
Oncogene
Viruses
Carcinogens
Random mutations
But what does ras
do in the cell??
But what does ras
do in the cell??
What do we want to know?
But what does ras
do in the cell??
Where does it do its work?
Ras is postranslationally modified
by addition of a lipid
--where will that put it?
Lipid modification
targets Ras to the plasma membrane
Farnesyltransferase inhibitors
offer a way of reducing ras activity
Farnesyltransferase inhibitors
offer a way of reducing ras activity
R115777
or lonafarnib
Phase II trials:
Leukemias (esp. AML/CML): Initial results promising
(also Advanced Multiple Myeloma)
Urothelial tract (with gemcitabine): in progress
Pancreatic cancer (with gemcitabine): no improvement
Colorectal cancer (with Irinotecan): just beginning
Small cell lung cancer (with Taxol): Discontinued.
What else could we ask about Ras?
Ras binds GTP
Is it a new type of kinase?
Lodish et al. Fig. 20-5
Ras is an enzyme
and hydolyzes GTP
Lodish et al. Fig. 20-5
Does this remind you
of another well studied signaling pathway
that was one of the first identified?
Lodish et al. Fig. 20-5
This allows Ras to act as
a molecular switch
Lodish et al. Fig. 20-5
The activity of Ras is regulated by
GEFs and GAPs
How is oncogenic ras different from the
normal proto-oncogene?
A new race starts
How is oncogenic ras different from the
normal proto-oncogene?
How is oncogenic ras different from the
normal proto-oncogene?
Ras is a key player in >50% of human tumors!
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
i.e. stuck in the ON state!
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