1. What Viruses Taught Us About Cancer
The story of Src
What Viruses Taught Us About Cancer

2. What Causes Cancer? - Random mutations (mistakes at the assembly line)
- Inherited mutations (pre-disposition)
- Viral infections
- Environmental factors (chemical; physical)
Viral infection as a cause for cancer is not common in humans, but it does exist (human papillomavirus can cause carcinoma of the uterine cervix)

3. Viruses cause cancer in animals
The key to our understanding of the molecular and cellular mechanisms of cancer in humans

4. Bacteria and viruses are infectious agents
Infection ?
If infected by a filtrate = a virus

5. Viruses use the cellular machinery for replication and protein synthesis

6. The Rous Sarcoma Virus (RSV)
A virus can transform a normal cell into a tumor
Nobel prize in Physiology or Medicine 1966
Peyton Rous

7. Chemicals can directly induce cancer
Carcinogens
Chemicals can directly induce cancer
1920s
Viral Infection Out
Chemical Induction In
Yamagiwa

8. RSV can transform cells in culture
30 Years Later: Rebirth of RSV research
RSV can transform cells in culture
RSV stock
Howard Temin
Immortality
Harry Rubin

9. Studying cancer at the cellular level
No contact inhibition on cell division

10. Changes in cell property and morphology
Normal
Cancer
Normal
Cancer

11. RSV can transform cells in culture
- RSV can induce cancer
- Cancer is a disease of malfunctioning cells rather than abnormally developing tissues
- Cancer can be studied relatively easily at the cellular level

12. The central dogma
DNA
Transcription
mRNA
Translation
Protein

13. The problem: RSV contains RNA, not DNA
Howard Temin
Nobel prize in physiology and medicine 1975
Temin: 1964
David Baltimore

14. One organism missed the lecture on the central dogma
The retrovirus

15. Mutant RSVs, mutated in the src sequence, fail to induce cancer
The viral genome of the normal chicken virus (ALV) contains 3 viral genes
RSV, a relative of ALV, contains an additional sequence: Src
Peter Duesberg
Mutant RSVs, mutated in the src sequence, fail to induce cancer

16. Conclusions
RSV transforms normal cells into cancerous cells by infecting them with the src gene
The src gene (and its product, the Src protein) is probably responsible for the other viral-associated cancers

17. Let’s label the src gene and follow its dynamics inside the host cell, after infection
Bishop and Varmus
Nobel prize in physiology and medicine 1989

18. The cellular src gene is highly conserved between different species
There is a cellular src that probably has healthy, normal roles in the cellular machinery
There is a viral src that promotes cancer
We distinguish between
c-src
v-src
(cellular)
(viral)

19. src was only the first one
Jennifer Harvey found a murine leukemia Virus, inducing sarcoma in rats: v-ras (later named H-ras)
Other viral transforming genes (14) showed similar patterns
c-src
v-src
(cellular)
(viral)
Proto-oncogene
Oncogene
v-src is similar, but NOT identical to c-src

20. How was v-src generated?
RSV exploited a kidnapped cellular gene
v-src is similar, but NOT identical to c-src

21. It is not all about mutations
Retroviruses can transform cells by altering gene expression (e.g. amplification)

22. So, what is src ?
Cells transformed by the v-src oncogene exhibit various cellular alterations
v-src affects a wide variety of cellular targets
The first BIG step:
using serum to immunoprecipitate the v-Src protein

23. Protein kinases and protein phosphatases add and remove phosphate groups from target proteins
Lodish et al. Fig. 20-5

24. Src is a Tyrosine Kinase
As a kinase, it can affect many cellular events
Figure 15-18a Molecular Biology of the Cell (© Garland Science 2008)

25. Normally, Src kinase intrinsic activity is low
What do we have here?
A _______ blot
These are proteins identified by antibodies that recognize phosphorylated tyrosine

26. Compare c-Src with conserved Src proteins
Src contains three domains that are shared with other proteins
Binds polyproline motifs
Phosphorylates
other proteins
Binds peptides phosphorylated on Tyr

27. Src is phosphorylated on its Tyr residue

28. Phosphorylated Tyr 527 inactivates Src
RCSB Protein databank

29. How is Src activated normally?
Martin, Nature Reviews MCB (2001)
- Dephosphorylation of Y527
- Configuration changes (e.g. SH2 binds to other proteins)

30. Recent work has provided a more detailed model of Src activation
Closed = OFF
Open = ON
Cowan-Jacob et al. Structure 13, (2005)

31. Cowan-Jacob et al. Structure 13, 861-871 (2005)
- Myristylation of Src to the cell membrane is the first step in its activation

32. And what about v-Src ?

33. The kidnapped cellular gene was only a partial gene

34. Summary - RSV and other retroviruses can transform cells into tumors
- Transformed cells exhibit changes in cell shape and behavior
- A viral oncogene is responsible for transformation
- Viral oncogenes are mutated versions of cellular proto-oncogenes
- The v-Src protein is a constitutively active tyrosin kinase

35. c- Src is a tyrosine kinase
What does it do in the cell?
What are its targets?

36. Identifying the targets of Src
Myristylation of Src is essential for transformation

37. p120 catenin: modulates cell-cell adhesion
Identifying The Targets of Src
Western blotting with anti-phosphotyrosine antibodies
V = v-Src transfected cells
2A/V = non-myristylated v-Src transfected cells
p120 catenin: modulates cell-cell adhesion
Reynolds et al. MCB (1989)

38. Identifying the targets of Src
Few Examples
- p120 catenin: modulates cell-cell adhesion
- Cortactin A: regulates actin polymerization
- Focal Adhesion Kinase: involved in cell-matrix interactions
Mike Schaller, ex-UNC

39. Src modulates both cell-cell and cell matrix adhesion
junctions
Cell-matrix junctions
Basal lamina

40. Epithelial cells secrete a special ECM called the basal lamina
Connective tissue

42. Integrins cluster into sites of contact with the ECM called focal adhesions

43. Focal adhesions are sites of intense protein tyrosine phosphorylation
Actin: Green
Phosphotyrosine: Red

44. Olga Krylyshkina

45. The ECM serves as a substrate for migrating cells
Revathi Ananthakrishnan 07

46. Albert Harris, Biology UNC
Matrix affects cell
Cells affect matrix
Albert Harris, Biology UNC
Pioneering the studies of forces exerted by cells on the matrix (e.g. cells can remodel the matrix)

47. focal adhesion proteins
Zoom into the focal adhesions
actin filaments
focal adhesion proteins
integrins
Plasma membrane

48. FAK is recruited by integrins to the membrane and is autophosphorylated
- Src binds to phophorylated FAK
- Src changes conformation and becomes active
- Src further phosphorylates FAK
- Src-FAK phosphorylate target proteins

49. Src and FAK act together to regulate other focal adhesion proteins
Src-FAK signals to regulate adhesion turnover
Src-FAK active = less adhesion, more migration

50. Src- FAK also promote cell survival and growth

51. Summary
- Viruses can act as infectious “cancer agents” via viral oncogenes
- Viral oncogenes are mutated versions of cellular proto-oncogenes
- The v-Src protein is a constitutively active tyrosin kinase, as a result of lack of Tyr at the C-terminus
- c-Src phosphorylates target proteins, including FAK
- FAK and c-Src act in the focal adhesions
- Focal adhesions are sites of cell-matrix interactions (both ways)

52. Rules of evidence How do we know this?
Src is localized at the right place and time
(correlative evidence)
src is sufficient
(gain-of-function evidence)
src is necessary
(loss-of-function evidence)

53. v-Src promotes adhesion dynamics and cell migration
focal adhesions
actin
normal cells
v-Src transformed cells
lose actin-membrane attachments
lose adhesion
Frame et al. Nature Review MCB (2002)

54. Rules of evidence How do we know this?
Src is localized at the right place and time
(correlative evidence)
src is sufficient
(gain-of-function evidence)
src is necessary
(loss-of-function evidence)

55. If Src is a critical regulator of cell adhesion, what happens to
an animal without any Src?
Knockout mice
Oliver Smithies
UNC
Nobel prize in physiology and medicine 2007

56. If Src is a critical regulator of cell adhesion, what happens to
an animal without any Src?
Knockout mice
- Defects in bone remodeling
- No lethality
Soriano et al. Cell (1991)

57. Ubiquitous expression: Src Yes Fyn
9 different murine Src family protein kinases compensate for each other
Ubiquitous expression:
Src Yes Fyn
How can we test their functions?

58. Klinghoffer et al. EMBO (1999)
src; fyn; yes triple mutant mice die at embryonic day 9.5 with multiple defects
Triple mutant
Wild-type
Klinghoffer et al. EMBO (1999)

59. Wound healing/Scratch assay

60. src; fyn; yes (SYF) triple mutant cells fail to migrate
Scratch assay

61. Rules of evidence How do we know this?
Src is localized at the right place and time
(correlative evidence)
src is sufficient
(gain-of-function evidence)
src is necessary
(loss-of-function evidence)

62. Figure 1

63. Table 1

64. Figure 2

65. Figure 3

66. Constitutively active Src
Src as a target for anti-cancer drugs
Dasatinib
Regulated Src
Constitutively active Src

67. Src as a target for anti-cancer drugs
50 clinical trials, 14 types of tumors
Usually, in combination with other drugs
dasatinib
bosutinib
saracatinib
Inhibit Src active site

68. Before we leave, what do we know?
Viruses kidnapped a normal proto-oncogene
During the “kidnapping”, the proto-oncogene became an oncogene
A new viral infection inserted an oncogene into the recipient, leading to cancer