Martin Simard Ph.D.Université Laval

Centre de Cancérologie (CHUQ)

MicroRNAs: from oncogenesis

comprehension to promising arsenal of

cancer therapies

MicroRNAs: from oncogenesis

comprehension to promising arsenal of

cancer therapies

•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 microRNAs: A new class of regulatory molecules

more than 1,000 different microRNAs in human

How microRNAs are produced?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

How microRNAs work? 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

miR-1-2

Precise expression profile Precise expression profile

Zhao et al, Nature 2005

Pena et al, Nature Methods 2009

miR-1-1

Stefani and Slack, Nature Reviews MCB 2008

Implication in cell differentiationImplication in cell differentiation

Precise expression profile: haematopoiesis Precise expression profile: haematopoiesis

Gangaraju and Lin, Nature Reviews MCB 2009

The loss of microRNAs lead to fatalityThe 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

microRNAs and Cancer microRNAs and Cancer

microRNAs are frequently located in altered genomic

regions associated to various cancers

Calin et al, PNAS 2004

microRNAs and Cancer microRNAs and Cancer

Different expression profile between healthy

and cancer tissue samples

Lu et al, Nature 2005

•Oncogenes

microRNAs and Cancer microRNAs and Cancer

Oncogene 2006

microRNA as Oncogene : miR-10bmicroRNA 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

•Oncogenes

•Tumour suppressors

microRNAs and Cancer microRNAs and Cancer

Oncogene 2006

microRNA as Tumour suppressor : let-7microRNA 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

microRNA as Tumour suppressor : miR-335 and miR-126microRNA 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

miR-34

BCL2 CDK4 CDK6CyclinE2

Cell proliferationSurvival

p53p53

He et al. Nature 2007 (+4)

Implication of the p53Implication of the p53

miR-34

BCL2 CDK4 CDK6CyclinE2

Cell proliferationSurvival

miR-16-1

miR-143

miR-145

p53p53

Suzuki et al. Nature 2009

He et al. Nature 2007 (+4)

Implication of the p53Implication of the p53

microRNAs and Cancer: more and more examples 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

Spizzo et al, Cell 2009

Kota et al. Cell, 2009

Decrease in tumour mass

microRNA as a Cancer therapy: miR-26a microRNA as a Cancer therapy: miR-26a

New therapies: Controlling microRNA expression New therapies: Controlling microRNA expression

Science, 2008

Controlling microRNA expression: different methodsControlling microRNA expression: different methods

Garzon et al, Nature Reviews Drug Discovery, 2010

Why microRNA are functionally important in primary cancers?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

microRNAs and Cancer: new biomarkers microRNAs and Cancer: new biomarkers

Nature Biotechnology 2008

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 microRNAs and Cancer: new biomarkers

British J. of Surgery, 2009

microRNAs and Cancer: new biomarkers to predict drug resistance

microRNAs and Cancer: new biomarkers to predict drug resistance

Clinical Cancer Research, 2009

Why microRNA are excellent biomarkers?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

Why microRNA are excellent biomarkers?Why microRNA are excellent biomarkers?

Weber et al, Clinical chemistry, 2010

Our microRNA profiling platformOur microRNA profiling platformTaqMan based quantitative RT-PCRTaqMan based quantitative RT-PCR

Our microRNA profiling platformOur microRNA profiling platform

•Extremely sensitive (from 10

cells)

•Great specificity

•No hybridization required

•Easily go from profiling to

validation

let-7c

let-7aUGA GGU AGU AGG UUG UAU AGU

Ulet-7b

UGA GGU AGU AGG UUG UGU GGU UUGA GGU AGU AGG UUG UAU GGU U

let-7e

let-7d AGA GGU AGU AGG UUG CAU AGU

UGA GGU AGG AGG UUG UAU AGU

•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 microRNAs are associated to various diseases

•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 summarizemicroRNAs: A new class of regulatory molecules

To summarizemicroRNAs: A new class of regulatory molecules

More than 17,000 microRNAs found in 142 species!

•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

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

us you

Translational research:What we can do as collaborators Translational research:What we can do as collaborators

Take home message Take home message

Stop thinking about DNA...

... think about RNA!

AcknowledgmentsAcknowledgments

Victor Ambros, U. Mass, Med. School

Gyorgy Hutvàgner, Dundee University

CollaboratorsCollaboratorsThe LabThe LabGuillaume

Jannot

Evelyne Rondeau

Samir Bouasker

Gabriel Bossé

Isabelle BanvilleMarie-Eve Boisvert

Dinshaw Patel, Sloan-Kettering

Nellie Giguère

Irfan Syed Bukhari

Alejandro Vasquez

Sandra PiquetMichael 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