Download Ras Part 1

Cellular Oncogenes
We made it to the 70s
Viral oncogenes paved the way
c-src
proto-oncogene
v-src
oncogene
Viral oncogenes paved the way
c-src
proto-oncogene
v-src
oncogene
The concept:
-Viruses kidnap a normal proto-oncogene
-During the “kidnapping”,
the mutated proto-oncogene 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
We now know one oncogene mutated in the progression
to malignancy is K-ras
Lodish
et al.
Fig. 24-6
How did we get there?
Onto the stage stepped a bright and ambitious
young assistant professor with a crazy idea
Bob Weinberg long before he wrote our textbook
But how can we identify 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 was 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
They got transformation using DNA from
chemically mutagenized 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 with DNA from 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 may
be dealing with a 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?
Generate a bacteriophage
genomic library
Search for the one human
gene among many mouse
genes using a humanspecific mobile element
as a probe
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
In 1981 there was no way to sequence 25 kilobases
The cloned gene is ~25,000 bp
In 1981 there was 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
Use Southern blot analysis to look for one
human gene in the otherwise mouse genome
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
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?
Where does it do it’s 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 thus
offered a way of reducing Ras activity
Clinicians then tried them in a variety of tumors
with activated Ras involvement
Tipifarnib
Or Lonafarnib
Examples of Phase II trials:
Leukemias (esp. AML/CML):
Metastatic breast cancer(with capecitabine):
Pancreatic cancer (with gemcitabine):
Ovarian cancer (with current 2 drug combo):
Neuroblastoma and Small cell lung cancer (with Taxol):
Sadly, it was largely an epic fail
Tipifarnib
Or Lonafarnib
Examples of Phase II trials:
Leukemias (esp. AML/CML)May have some efficacy
Metastatic breast cancer(with capecitabine): no significant improvement
Pancreatic cancer (with gemcitabine): no improvement
Ovarian cancer (with current 2 drug combo): no effect
Neuroblastoma and Small cell lung cancer (with Taxol): Discontinued.
We now know parallel pathways can
add related lipids to Ras,
Restoring membrane targeting
Tipifarnib
Or Lonafarnib
What else could we ask about Ras?
Scientists found 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
2012 Nobel Prize in Chemistry!
Nobel Prize.org
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
They made chimeras to identify the region
of Ras that confers activity
They made chimeras to identify the region
of Ras that confers activity
One amino acid!!!!
Ras is a key player in >50% of human tumors!
Table 4.2 The Biology of Cancer (© Garland Science 2007)
A note for the future—Ras is mutated in
<5% of human breast cancers—we’ll see why later
Table 4.2 The Biology of Cancer (© Garland Science 2007)
The G12V mutation prevents endogenous
and GAP-stimulated GTPase activity
Thus 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