1. Genomic signatures in colorectal cancer
“Highlights in Metastatic Colorectal Cancer”
Roma, 4 marzo 2011
Genomic signatures in colorectal cancer
Mirco Menigatti MD, PhD
Institute of Molecular Cancer Research
University of Zurich (Switzerland)

2. Colorectal cancers Hereditay Familial Sporadic
adapted from Kemp Z et al. Hum Mol Genet Oct 1;13 Spec No 2:R

3. The colorectal tumor progression
(HNPCC)
Grady WM, Carethers JM. Gastroenterology Oct;135(4):

4. Genomic instability   Chromosomal instability (85%)
Alterations in mitotic-spindle checkpoint
and sister-chromatid separation pathways. 
Chromosomal instability (85%)
Loss of function of the DNA Mismatch Repair system (HNPCC, epigenetic silencing of MLH1). 
Microsatellite instability (15%)

5. A genetic model for colorectal tumorigenesis.
Fearon ER, Vogelstein B. Cell Jun 1;61(5):759-67

6. The genomic landscape of human colorectal cancers
analysis of exons representing 20,857 transcripts from 18,191 genes
Wood LD et al. Science Nov 16;318(5853):

7. Somatic mutations Drivers:
causally involved in the neoplastic process and positively selected for during tumorigenesis.
Passengers:
no positive or negative selective advantage to the tumor.
Colorectal cancers  a median 76 genes altered by point mutations.
Only 14 can be considered as drivers.

8. Copy number changes Amplifications Homozygous deletions
Colorectal cancers have a median 9 genes altered by a major copy number change
Amplifications
(i.e. MYC, EPPB9, EGFR)
Homozygous deletions
(i.e. PTEN, TP53, MAP2K4, SMAD2)
Leary RJ et al. Proc Natl Acad Sci U S A Oct 21;105(42):

9. Alterations of signaling pathways
WNT
TGFβ / SMAD
Notch
Hedgehog
RAS
EGFR
PI3K/Akt

10. Cancer phenotype
Genetic and Epigenetic alterations

11. Epigenetics
Historically, the word “epigenetics” was used to describe events that could not be explained by genetic principles.
Waddington, C.H. (1942). Endeavour 1, 18–20.

12. Goldberg AD et al Cell. 2007 Feb 23;128(4):635-8.
EPIGENETICS The study of any potentially stable and inheritable change in gene expression or cellular phenotype that occurs in the absence of changes in Watson-Crick base-pairing of DNA.
histone modifications
DNA methylation
microRNAs
Long non coding RNAs
Goldberg AD et al Cell Feb 23;128(4):635-8.

13. DNA methylation
In mammals, nearly all DNA methylation occurs on cytosine bases that are located 5' to a guanosine in a CpG dinucleotide (CpG sites)

14. DNA methylation Silencing of repetitive and centromeric sequences
It is the best characterized chemical modification of chromatin.
It plays a role in many cellular processes :
Silencing of repetitive and centromeric sequences
X chromosome inactivation in female mammals
Mammalian imprinting

15. ALTERATED LEVELS OF DNA METHYLATION IN TUMORS
HYPOMETHYLATION  within the coding regions of genes are
present CpG sites with low density. Most of them are normally methylated 
Hypomethylation,during carcinogenesis, may lead to chromosomal instability
HYPERMETHYLATION  de novo methylation of CpG islands present in ~60% of human gene promoters which are normally found unmethylated 
Silencing of gene expression (depending on the density of promoter methylation)

16. Transcription Silencing
HYPERMETHYLATION
Cell cycle control → p16
Repair of DNA damage → MLH1
Apoptosis → Dap kinase
Tumor-cell invasion → TIMP3
Growth-factor response → ER

17. MGMT
CpG unmet
CpG met
Menigatti et al Oncogene 2009

18. Histone changes
adapted from Sparmann A, van Lohuizen M. Nat Rev Cancer. 2006

19. microRNAs
Iorio, M. V. et al. J Clin Oncol; 27:

20. Mechanisms of microRNA (miR) regulation
Iorio, M. V. et al. J Clin Oncol; 2009

21. GENETIC ALTERATIONS CAUSING EPIGENETIC CHANGES IN CANCER
PML-RAR fusion protein in acute promyelocytic leukemias
induces RARß2 gene promoter hypermetylation and silencing
by recruiting DNA methyltransferases to its promoter
(Di Croce et al. Science, 2002)

22. EPIGENETIC CHANGES CAUSING GENETIC ALTERATIONS IN CANCER
MLH1 promoter hypermethylation 
Mismatch repair deficiency
Mutation in genes with repetitive
sequences
(BAX, TGFß RII, etc.)
MGMT promoter hypermethylation 
No removal of G alkyl adducts
G to A mutations in oncogenes
(KRAS) and tumor suppressor genes (p53)

24. Microarray transcription profiling
32 normal mucosal samples
32 adenomas
level of expression (blue, low; red, high)

25. PTPRR mRNA levels Normalized intensity Normal Mucosa (no=32) Polypoid
adenomas
(n=32)
Colorectal
cancers
(n=25)
Colon cancer
cell lines
(n=18)

26. PTPRR
Encodes the classical transmembrane protein-tyrosine phosphatase (PTP) known as PTP, receptor type, R.
Reversible tyrosine-specific phosphorylation of cellular proteins is a key signalling mechanism used to evoke essential cell decisions such as proliferation and differentiation and its proper regulation depends on the balanced activities of PTPs and protein tyrosine kinases (PTKs).

27. Re-activation of PTPRR expression

29. Histone code H3K9ac H3K27me3 H3K9me3 Colo205 (PTPRR silenced)
Input %
Colo205 (PTPRR silenced)
SK-N-SH (expressing PTPRR)
H3K9ac
H3K27me3
H3K9me3

31. in progress

32. Stem cells
miRNAs

33. CRC stem-like cell lines CD133+ Angelo Vescovi, University of Milano, Italy

34. Expressed only in CRC stem-like
cell lines CD133+
Silenced only in CRC stem-like
cell lines CD133+

35. Translational Epigenetics
Early detection
Response to therapeutics
Epigenetic therapy

36. Non invasive tests: serum, bronchoalveolar lavage , urine and stool
Early detection
Non invasive tests: serum, bronchoalveolar lavage , urine and stool
Aberrant methylation of some gene promoters is more common and easier to detect than mutations 
sensitivity/specificity: 92% / 86%
Stool-based analyses of a combination of DNA methylation markers achieving at least
85% sensitivity for cancer, 50% sensitivity for pre-cancer with 90% specificity.

37. Response to therapeutics
Patients with MGMT methylation  median survival of 21.7 months (15.3 months without temozolomide therapy).
Patients without MGMT methylation  median survival of 12.7 months (11.8 months without temozolomide therapy).

38. Epigenetic therapy
Taby R, Issa JP. CA Cancer J Clin Nov-Dec;60(6):

39. Readings Jones PA, Baylin SB. The epigenomics of cancer.
Cell Feb 23;128(4):
Esteller M. Epigenetics in cancer.
N Engl J Med Mar 13;358(11):
Brena RM, Costello JF. Genome-epigenome interactions in cancer.
Hum Mol Genet Apr 15;16 Spec No 1:R
Jiricny J, Menigatti M. DNA Cytosine demethylation: are we getting close?
Cell Dec 26;135(7):

40. LINKS The Epigenome Network: www.epigenome-noe.net
Epigenetics society:
DNA Methylation in Cancer:
EMBOSS CpGPlot :

41. TOP EUROPEAN
United Kingdom
Germany 
Netherlands 
Switzerland

42. GRAZIE ROMA