3. The story of APC and the Wnt pathway

5. The story of APC and the Wnt pathway

6. Signal Transduction Pathways
Important for development and homeostasis.
All involved in Cancer.
RTK
Wnt
TGF-β
Hedgehog
Notch

7. 2007 Estimated US Cancer Deaths*
Men 289,550
Women 270,100
Lung & bronchus 31%
Prostate 9%
Colon & rectum 9%
Pancreas 6%
Leukemia 4%
Liver & intrahepatic 4% bile duct
Esophagus 4%
Urinary bladder 3%
Non-Hodgkin % lymphoma
Kidney 3%
All other sites %
26% Lung & bronchus
15% Breast
10% Colon & rectum
6% Pancreas
6% Ovary
4% Leukemia
3% Non-Hodgkin lymphoma
3% Uterine corpus
2% Brain/ONS
2% Liver & intrahepatic bile duct
23% All other sites
ONS=Other nervous system.
Source: American Cancer Society, 2007.

8. Colon cancer is the second leading cause of cancer deaths in the U.S.
(> 50,000 deaths per year)
150,000 cases diagnosed per year
(i.e., your chances are 1/18)

9. Colorectal cancer Early detection is the key
>90% 5 year survival with early detection
60% if it has spread locally
<10% if it has metastasized
Polyps, the first stage
In tumor development
Data from ACS
Figure 2.15a The Biology of Cancer (© Garland Science 2007)

10. Colorectal cancer treatment
1. Surgical removal of tumor
This can be followed by either
Radiation or chemotherapy (fluorouracil)
3. Chemotherapy also used to
slow the progress of metastatic disease
Data from ACS

11. Colon cancer genetics 95% of cases sporadic (no genetic history)
HNPCC 3% of all cases.
Defects in mismatch repair.
Familial adenomatous polyposis (FAP)
1% of all colon cancers

12. Familial Adenomatous Polyposis (FAP)
polyps by age 30
Figure The Biology of Cancer (© Garland Science 2007)

13. Inheriting FAP-related mutations dramatically increases
your risk of developing colorectal cancer.
93% colorectal cancers by age 50
0.2% colorectal cancers by age 50
100% colorectal cancers lifetime risk
6% colorectal cancers lifetime risk

14. Until recently the only treatment was to surgically remove the colon
Treatment with an aspirin-like drug
called celecoxib (a COX2 inhibitor)
led to a 25% reduction in polyp number ,
and the remaining polyps shrank.
The hope is this will allow doctors to
delay surgery till later in life
   
Steinbach et al.
New England Journal of Medicine 342, 1946 (2000)

15. Figure 7.23 The Biology of Cancer (© Garland Science 2007)

16. Adenomatous Polyposis Coli (APC)
Not to be confused with:
APC (anaphase-promoting complex)
APC (antigen-presenting cell)
nor with:
The American Pie Council (APC) committed to preserving America's pie heritage
Armoured Personnel Carrier

17. We have positioned and cloned a gene
What is the next step?
looks like……..
NOTHING!!

18. but cancer involves two mutated copied
mutated in FAP
and in 70% of sporadic
cases of colon cancer
dominant inheritance
but cancer involves two mutated copied
tumor suppressor gene
APC mutations are
the earliest event
in the development
of the tumor

19. Tell me who are your friends and I will tell you who you are.
If we don’t know what APC is doing, let’s find binding partners
APC
cell extract

20. First, immunoprecipitation
Thermo Scientific

21. co-immunoprecipitation
Thermo Scientific

22. Fishing with APC ? first, immunoprecipitation APC
Colon cancer cell line WT ?
first,
immunoprecipitation
APC

23. co-immunoprecipitation
Fishing with APC
Colon cancer cell line WT
co-immunoprecipitation
p95= -catenin

24. What is -catenin?

25. Drosophila is a segmented animal.
The different parts and organs in the body are derived from a total of 14 embryonic segments.

26. Each segment is composed of rows of cells.

28. Each segment is polarized in the anterior-posterior axis.
The cuticle in each segment has a hairy anterior part and a naked posterior part.
In other words, cells from the different halves of the segment have different fates.

29. Genetics is the Key Tool for Understanding Drosophila Development
Christiane Nusslein-Volhard
Eric Wieschaus
Nobel prize in physiology and medicine 1995

30. distinctive structures
The wild-type body
is segmented
and each segment
has a unique identity
and thus produces
distinctive structures

31. The Heidelberg screen 2 young scientists 1 microscope 1 year
26,978 flies
139 new genes
Wieschaus and Nüsslein-Volhard removed single genes (random mutagenesis) and looked for effects on the body plan

32. In some of the mutants: no naked cuticle
wild type
mutant

33. The name of the mutant: armadillo

34. 1. adherens junctions (wait till Thursday..)
armadillo was cloned
armadillo = -catenin
1. adherens junctions (wait till Thursday..)
2. cytoplasmic/nuclear
Dr. Mark Peifer

35. -catenin and TCF form a bipartite transcription factor

36. armadillo is not the only mutant without a naked cuticle
wild type
arm
(and others)

37. -catenin and TCF form a bipartite transcription factor
-catenin is phosphorylated by GSK-3 for degradation

38. levels of Armadillo/  -catenin rise dramatically
In the absence of APC,
levels of Armadillo/  -catenin rise dramatically
wild-type
APC mutant
McCartney, Dev 06
Rosin-Arbesfeld, Nature 00

39. Mutation Cluster Region
APC is a complex protein
bind to Armadillo
15 AA
rpts
20 AA
rpts
Mutation Cluster Region
bind to Axin

40. armadillo is not the only mutant without a naked cuticle
wild type
arm
(or wingless)

41. Signal Transduction Pathways
Important for development and homeostasis.
RTK
Wnt (wingless)
TGF-β
Hedgehog
Notch

42. Wingless/Wnt regulates Armadillo/  -catenin stability and promotes specific cell fates.

43. Back to Colon Cancer

44. away from the crypt, they
A quick look at colon architecture
differentiated
cells
villus
As cells migrate
away from the crypt, they
differentiate.
proliferating
cells
crypt
Wnt

45. The genes turned on by Wnt signaling are expressed in crypts
and those that are turned off are
expressed in villi
Gene turned ON by Wnt
Gene turned OFF by Wnt
Hans Clevers

46. The effect on p21 expression is indirectly mediated by
the transcription factor c-myc.
Transcription of the myc gene is directly regulated
By TCF/beta-catenin.
As cells migrate
away from the crypt, they
differentiate.

47. Let’s put this together to form a picture of normal colon biology
and how it is perturbed by APC mutations
loss of function
As cells migrate
away from the crypt, they
differentiate.
Stromal cells send Wnt signals.