Genomic signatures in colorectal cancer

1. “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. 2004 Oct 1;13 Spec No 2:R177-85.

3. The colorectal tumor progression (HNPCC) Grady WM, Carethers JM. Gastroenterology. 2008 Oct;135(4):1079-99

4. Genomic instability 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. 1990 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. 2007 Nov 16;318(5853):1108-13.

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 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. 2008 Oct 21;105(42):16224-9

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. EPIGENETICSThe 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 alCell. 2007 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 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 thecoding regionsof genes are present CpG siteswithlow density.Most of them arenormally methylated  Hypomethylation,during carcinogenesis, may lead tochromosomal instability HYPERMETHYLATIONde novo methylation ofCpG islandspresent in ~60% ofhuman gene promoters which are normally found unmethylated  Silencing of gene expression(depending on the density of promoter methylation)

16. Transcription HYPERMETHYLATION Silencing 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; 27:5848-5856 2009

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 transmembraneprotein-tyrosine phosphatase(PTP) known as PTP, receptor type, R. • Reversibletyrosine-specific phosphorylationof 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 ofPTPsand protein tyrosine kinases(PTKs).

27. Re-activation of PTPRR expression

29. Histone code Input % SK-N-SH (expressing PTPRR) Colo205 (PTPRR silenced) H3K27me3 H3K9ac 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. 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. 2010 Nov-Dec;60(6):376-92.

39. Readings Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007 Feb 23;128(4):683-92. Esteller M. Epigenetics in cancer. N Engl J Med. 2008 Mar 13;358(11):1148-59 Brena RM, Costello JF. Genome-epigenome interactions in cancer. Hum Mol Genet. 2007 Apr 15;16 Spec No 1:R96-105. Jiricny J, Menigatti M. DNACytosine demethylation: are we getting close? Cell. 2008 Dec 26;135(7):1167-9.

40. LINKS The Epigenome Network: www.epigenome-noe.net Epigenetics society: www.dnamethsoc.com DNA Methylation in Cancer: www.mdanderson.org/departments/methylation EMBOSS CpGPlot : www.ebi.ac.uk/emboss/cpgplot

41. TOP EUROPEAN United Kingdom Germany  Netherlands  Switzerland

42. GRAZIE ROMA