1. Martin Simard Ph.D. Université Laval Centre de Cancérologie (CHUQ) MicroRNAs: from oncogenesis comprehension to promising arsenal of cancer therapies

2. Small non-coding RNAs (21 to 23 nucleotides long) First discovered in Caenorhabditis elegans Found in nearly all eukaryotes Act “negatively” in gene expression and control cellular timing Recent estimation support that more than 60% of the human coding genome is directly regulated by microRNAs microRNAs: A new class of regulatory molecules more than 1,000 different microRNAs in human

3. How microRNAs are produced? Mainly produced by RNA polymerase II. 1st maturation in the nucleus 2nd maturation in the cytoplasm Active complex: miRISC Nature Reviews MCB, 2008

4. How microRNAs work? Partial complementarity with 3’UTR regions (position 2 to 8 critical) Cooperative effect Abrogate protein synthesis One microRNA may regulate up to 100 different genes! Nature Reviews MCB, 2008

5. miR-1-2 Precise expression profile Zhao et al, Nature 2005Pena et al, Nature Methods 2009 miR-1-1

6. Stefani and Slack, Nature Reviews MCB 2008 Implication in cell differentiation

7. Precise expression profile: haematopoiesis Gangaraju and Lin, Nature Reviews MCB 2009

8. The loss of microRNAs lead to fatality Zhao et al, Cell 2007 Ventura et al, Cell 2008 Loss of miR-1-2 leads to overproduction of muscle cells Loss of miR-17- 92 cluster is embryonic lethal

9. microRNAs and Cancer microRNAs are frequently located in altered genomic regions associated to various cancers Calin et al, PNAS 2004

10. microRNAs and Cancer Different expression profile between healthy and cancer tissue samples Lu et al, Nature 2005

11. Oncogenes microRNAs and Cancer Oncogene 2006

12. microRNA as Oncogene : miR-10b Overexpression increases angiogenesis Induces metastasis formation Correlation between miR-10b overexpression and metastatic tumours caused by breast cancer Ma et al, Nature 2007

13. Oncogenes Tumour suppressors microRNAs and Cancer Oncogene 2006

14. microRNA as Tumour suppressor : let-7 let-7 level is altered in lung cancer let-7 controls RAS expression HMGA2 is another target of let-7 Johnson et al, Cell 2005 Mayr et al, Science 2007

15. microRNA as Tumour suppressor : miR-335 and miR-126 Re-establishing their expressions diminish the metastasis formation miR-335 controls cell migration miR-126 controls cell proliferation Tavazoie et al, Nature 2008

16. miR-34 BCL2 CDK4 CDK6 CyclinE2 Cell proliferation Survival p53 He et al. Nature 2007 (+4) Implication of the p53

17. miR-34 BCL2 CDK4 CDK6 CyclinE2 Cell proliferation Survival miR-16-1 miR-143 miR-145 p53 Suzuki et al. Nature 2009 He et al. Nature 2007 (+4) Implication of the p53

18. microRNAs and Cancer: more and more examples Mutation or epigenetic changes can lead to: Deletion of microRNA Epigenetic silencing of microRNA locus Point mutation affecting a microRNA or microRNA precursor Genomic amplification or translocation of microRNA locus Loss of epigenetic silencing of microRNA locus Point mutation in the microRNA targets Rearrangement of 3’UTR miR-15a,-miR- 16-1 miR-29, miR- 203 miR-15a,-miR- 16-1 HMGA- 2 miR-17~92, miR- 21

19. Spizzo et al, Cell 2009

20. Kota et al. Cell, 2009 Decrease in tumour mass microRNA as a Cancer therapy: miR-26a

21. New therapies: Controlling microRNA expression Science, 2008

22. Controlling microRNA expression: different methods Garzon et al, Nature Reviews Drug Discovery, 2010

23. Why microRNA are functionally important in primary cancers? There are tumours-specific microRNA signatures which accurately distinguish different sub-types of cancers Modulation of microRNAs in cancer cell lines can directly regulates fundamental behaviours of cancer cells such as proliferation and apoptosis Many microRNAs deregulated in cancers have been shown to control oncogenes, tumour suppressors and signalling pathway components as direct targets

24. microRNAs and Cancer: new biomarkers Nature Biotechnology 2008

25. Using ovarian cancer as a model, we demonstrate that expression of let-7 and HMGA2 is a better predictor of prognosis than classical markers such as E-cadherin, vimentin, and Snail. These data identify loss of let-7 expression as a marker for less differentiated cancer. PNAS, 2007 microRNAs and Cancer: new biomarkers British J. of Surgery, 2009

26. microRNAs and Cancer: new biomarkers to predict drug resistance microRNAs and Cancer: new biomarkers to predict drug resistance Clinical Cancer Research, 2009

27. Why microRNA are excellent biomarkers? Extremely stable in fluids as well as on formalin- fixed paraffin-embedded tissue Expression profile correlates well between fresh and formalin-fixed paraffin-embedded samples Resistant to degradation

28. Why microRNA are excellent biomarkers? Weber et al, Clinical chemistry, 2010

29. Our microRNA profiling platform TaqMan based quantitative RT-PCR

30. Our microRNA profiling platform Extremely sensitive (from 10 cells) Great specificity No hybridization required Easily go from profiling to validation let-7c let-7a UGA GGU AGU AGG UUG UAU AGU U let-7b UGA GGU AGU AGG UUG UGU GGU U UGA GGU AGU AGG UUG UAU GGU U let-7e let-7dAGA GGU AGU AGG UUG CAU AGU UGA GGU AGG AGG UUG UAU AGU

31. Viral infections - Viruses encodes microRNAs that target viral mRNAs to regulate various stages of the viral life cycle - Viral microRNAs suppress expression of specific host genes - Viral infections induce expression of host microRNAs that inhibits expression of cellular genes - Upon viral infections, host cells express specific microRNA that suppress viral mRNA expression Cardiac, immune, neurological and metabolic disorders microRNAs are associated to various diseases

32. Small non-coding RNAs (21 to 23 nucleotides long) First discovered in Caenorhabditis elegans Found in nearly all eukaryotes microRNA as new biomarkers Modulation of microRNA expression: new therapeutic strategy To summarize microRNAs: A new class of regulatory molecules To summarize microRNAs: A new class of regulatory molecules More than 17,000 microRNAs found in 142 species!

33. What are the targets of microRNAs? How microRNA expression is regulated? How microRNAs regulate gene expression? Which are the cellular factors implicated in the microRNA pathway? Fundamental research: Questions to be answered Fundamental research: Questions to be answered

34. A collaboration with Sebastien Bonnet Journal of Experimental Medicine, in revision Implication of miR-204 in pulmonary arterial hypertension microRNA profiling from tissues Identification of microRNA targets Functional test in animal models usyou Translational research: What we can do as collaborators Translational research: What we can do as collaborators

35. Take home message Stop thinking about DNA...... think about RNA!

36. Acknowledgments Victor Ambros, U. Mass, Med. School Gyorgy Hutvàgner, Dundee University Collaborators The Lab Guillaume Jannot Evelyne Rondeau Samir Bouasker Gabriel Bossé Isabelle Banville Marie-Eve Boisvert Dinshaw Patel, Sloan- Kettering Nellie Giguère Irfan Syed Bukhari Alejandro Vasquez Sandra Piquet Michael Hengartner, Zurich University Eric Miska, Cambridge University Craig Mello, U. Mass Med. School Johannie Ducharme Sébastien Bonnet, Université Laval Ivan Robert Nabi, UBC Jean-Yves Masson, Université Laval Eric Paquet, CRC