1. Cellular Oncogenes

2. We made it to the 70s

3. Viral oncogenes paved the way
c-src
v-src
proto-oncogene
oncogene

4. Viral oncogenes paved the way
c-src
v-src
proto-oncogene
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 4

5. no known viral tumors in humans
However….
At the time, there were
no known viral tumors in humans

6. We ultimately want to know the cause of
human diseases like colon cancer,
rather than curing chicken sarcomas

7. We now know one oncogene mutated in the progression
to malignancy is K-ras
Lodish
et al.
Fig. 24-6

8. How did we get there? 8

9. Bob Weinberg long before he wrote our textbook
Onto the stage stepped a bright and ambitious
young assistant professor with a crazy idea
Bob Weinberg long before he wrote our textbook 9

10. But how can we identify oncogenes that are not viral oncogenes?

11. Add DNA from carcinogen-treated cell?
But how can we find oncogenes that are not viral oncogenes?
Add DNA from carcinogen-treated cell? 11

12. First you need a way
to get DNA into cells
How would you do that?

13. DNA can be “transfected” into cells using Calcium Phosphate

14. As you know, oncogenes relieve contact inhibition.
This can be visualized in culture
by “focus formation”
Transfect with oncogene
J Biol Chem : Fiordalisi et al.
J Virol : Yoshioka et al.

15. One can then test if the cells induce tumors

16. But it was a crazy idea
1. you need to find
the right needle in
a very large haystack
What if you need to find
two or more needles??

17. Chiaho Shih
Bob Weinberg

18. Maybe it wasn’t So crazy after all! Soon everybody was trying it!
Chiaho Shih
Bob Weinberg 18

19. a focus after transfection
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

20. It’s time to move on- the 80s

21. Could this work with DNA from human tumors?
Transfect DNA from human cancer cell lines
Human Cancer Cell Line
Chiaho Shih
Bob Weinberg

22. Somewhere here, among the normal mouse genes, we have a human oncogene
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

23. How to find a needle in a haystack
Figure 4.8 The Biology of Cancer (© Garland Science 2007)

24. 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 human- specific mobile element
as a probe
Lodish et al. Fig. 24-4

25. The Race is ON Wigler lab Barbacid lab Weinberg lab
December 1981
Barbacid lab
Weinberg lab
An Oncogene is Cloned
From a Human Tumor !!
But what does it encode?

26. 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

27. 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

28. What probe should we use ?
Use Southern blot analysis to look for one human gene in the otherwise mouse genome
What probe should we use ?

29. Just go through the viral oncogenes
one by one
mouse cells
human cells
human RAS
mouse RAS
Der et al. PNAS 82
Channing Der, now at UNC

30. transformed by a human oncogene
The transforming oncogene is Ras
Cellular oncogenes = Viral oncogenes
mouse cells
human cells
Mouse cells
transformed by a human oncogene
human RAS
mouse RAS
Der et al. PNAS 82
Channing Der, UNC 30

31. c-ras
v-ras
(cellular )
(viral)
Proto-oncogene
Oncogene
Carcinogens
Random mutations

32. But what does Ras
do in the cell??

33. But what does Ras do in the cell??
What do we want to know?

34. Where does it do it’s work?

35. Ras is postranslationally modified --where will that put it?
by addition of a lipid
--where will that put it?

36. targets Ras to the plasma membrane
Lipid modification
targets Ras to the plasma membrane

37. Farnesyltransferase inhibitors thus
offered a way of reducing Ras activity

38. 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):

39. 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.

40. We now know parallel pathways can add related lipids to Ras,
Restoring membrane targeting
Tipifarnib
Or Lonafarnib

41. What else could we ask about Ras?

42. Scientists found Ras binds GTP Is it a new type of kinase?
Lodish et al. Fig. 20-5

43. Ras is an enzyme
and hydolyzes GTP
Lodish et al. Fig. 20-5 43

44. of another well studied signaling pathway
Does this remind you
of another well studied signaling pathway
that was one of the first identified?
Lodish et al. Fig. 20-5 44

45. 2012 Nobel Prize in Chemistry!
Nobel Prize.org 45

46. This allows Ras to act as
a molecular switch
Lodish et al. Fig. 20-5 46

47. The activity of Ras is regulated by
GEFs and GAPs

48. How is oncogenic ras different from the normal proto-oncogene?
A new race starts

49. They made chimeras to identify the region of Ras that confers activity

50. They made chimeras to identify the region of Ras that confers activity

52. One amino acid!!!!

53. Ras is a key player in >50% of human tumors!
Table 4.2 The Biology of Cancer (© Garland Science 2007)

54. 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)

55. The G12V mutation prevents endogenous and GAP-stimulated GTPase activity

56. Thus the Ras oncogene is constitutively active
i.e. stuck in the ON state!
G12V
Lodish et al. Fig. 20-5

57. Why would constitutively active Ras lead to cancer ?
How does Ras act in our body, in vivo ?
From cell culture to model organisms