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

2. Caenorhabditis elegans Drosophila melanogaster
Model organisms teach us about ourselves
Mus musculus
Xenopus laevis
Caenorhabditis elegans
Drosophila melanogaster

3. 5 Key Signal transduction pathways
are critical for development and homeostasis
All play critical roles in Cancer

4. 5 Key Signal transduction pathways
Critical for development and homeostasis
All play critical roles in Cancer
RTK- Ras (me)
Wnt (me)
TGF-ß (you)
Hedgehog (you)
Notch (you) 4

5. We’ll first look at the RTK=
Receptor tyrosine kinase pathway
Src=non-receptor tyrosine kinase
RTKs

6. We’ll start by walking through the RTK pathway and then talk about how we got to this knowledge
RTKs

7. In the absence of Ligand with an poorly active kinase
RTKs are monomers
with an poorly active kinase

8. Ligand binding “activates” RTKs
by dimerization
Lodish et al. Fig

9. RTKs are their own substrates-- i.e., they autophosphorylate

10. Remember SH2 domains?
What did they bind??

11. SH2 domains allow “effector” proteins to bind activated receptors

12. One adaptor with an SH2 domains is Grb2
It also has SH3 domains--what do they bind?

13. One adaptor with an SH2 domains is Grb2
It also has SH3 domains--what do they bind?
Ras 13

14. Step by step--think dominos
Ras
Ras

15. Remember-Ras is anchored to the membrane through a lipid

16. And SOS is a GEF--remember them??

17. Each activated protein activates the next
Figure Molecular Biology of the Cell (© Garland Science 2008)

18. Each activated protein activates the next
MAPK/ERK enters nucleus
Figure Molecular Biology of the Cell (© Garland Science 2008) 18

19. Each activated protein activates the next
MAPK/ERK enters nucleus
Figure Molecular Biology of the Cell (© Garland Science 2008) 19

20. Each activated protein activates the next
MAPK/ERK enters nucleus
Figure Molecular Biology of the Cell (© Garland Science 2008) 20

21. The RTK pathway

22. We made it to the 90s 10 years-old Britney Spears
Travolta is still dancing
A new graduate from Harvard
Kurt Cobain

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

24. Caenorhabditis elegans Drosophila melanogaster
Model organisms teach us about ourselves
Mus musculus
Xenopus laevis
Caenorhabditis elegans
Drosophila melanogaster

25. The eye of a fly: One Key to learning how Ras and RTKs work
Lodish et al. Fig

26. Did you say flies?

27. Did you
say flies?

28. This is all
I need to know

29. But wait--flies helped us understand yhe single most important human
oncogene
Lodish et al. Fig

30. I told you the RTK pathway is key
in MANY developmental decisions

31. Each ommatidium contains all the cells needed to see the world
Including the eight photoreceptors

32. These cells choose fate one by one,
each telling the next what fate to adopt

33. sevenless mutants lack an R7
photoreceptor
Wild-type
sevenless mutant

34. Sevenless encodes an RTK!
Lodish et al. Fig

35. To get a cellular response,
There is a threshold level of pathway activity
R7 present
sev pathway activity
sev threshhold
R7 absent
wild type sev sevts sevts sevts; enh*/+
22.7o C o C o C
Gian Garriga

36. Scientists figured out how to tune RTK activity
using a temperature sensitive mutant
R7 present
sev pathway activity
sev threshhold
R7 absent
wild type sev sevts sevts sevts; enh*/+
22.7o C o C o C
Gian Garriga

37. They then looked for mutants In other genes that would drop
Pathway activity below the threshold
R7 present
sev pathway activity
sev threshhold
R7 absent
wild type sev sevts sevts sevts; enh*/+
22.7o C o C o C
Gian Garriga

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

39. Wow--Ras, the adapter Grb2 and a GEF are all in the RTK pathway!
Sos
Grb2
Ras

40. Further, Ras acts downstream of
the RTK Sevenless
Lodish et al. Fig

41. The signal transduction pathway
should look familiar
Grb2
Alberts et al. Fig

42. Because that’s how we
figured it out!
Grb2
Alberts et al. Fig

43. Flies were bad enough,
but worms!?
Caenorhabditis elegans

44. Luckily the Nobel Committee
thinks they are cool
Caenorhabditis elegans 44

45. Nobel Prize lineage and programmed cell death GFP RNAi
Physiology and Medicine 2002
Bob Horvitz
John Sulston
Sydney Brenner
Physiology and Medicine 2006
Chemistry 2008
GFP
RNAi
Andy Fire
Craig Mello
Marty Chalfie

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

47. 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

48. Cell Induction The AC signals the VPCs to adopt vulval fates
Only 3 VPCs will actually form the vulva

49. what proteins are required
How can we figure out
what proteins are required
to build a vulva?

50. GENETICS!

51. Mutants with no vulval signaling
wild type
No vulva induction
Mutants with no vulval signaling
vulvaless (Vul)

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

53. 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)

54. One key paper: The screen for Vul and Muv mutants
some examples
Bob Horvitz
Vul
Muv
let-23
lin-15
Nobel Prize
physiology and medicine 2002
lin-3
let-60

55. One key paper: The screen for Vul and Muv mutants
some examples
Bob Horvitz
Vul
Muv
let-23
lin-15
Nobel Prize
physiology and medicine 2002
lin-3
let-60
Next step: cloning and sequencing the genes

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

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

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

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

60. First evidence that Ras has an in vivo role as part of the RTK pathway

61. The Ras gain-of-function mutation =
Glycine 13 Glutamine

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

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

64. of the proteins in the pathway?
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 Muv phenotype] 64

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

66. This worked GREAT!
lin-15

67. 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

68. The RTK-Ras pathway also offers drug targets for cancer treatment
Alberts et al. Fig 68

69. 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) 69

70. 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)
And radioactive iodine resistant thyroid cancer (Nov. 2013) 70

71. Or how the drug company sells it71

72. 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) 72

73. The results were initially amazing

74. But….
We’ll come
back to this

75. Summary
- Cellular oncogenes = viral oncogenes

76. Summary - Cellular oncogenes = viral oncogenes
- Ras, as one of these genes, encodes a small GTPase, acting as a molecular switch 76

77. 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 77

78. 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 78