Webinar Series miRNAs and Cancer · Pre-miRNA (hairpin precursor) Drosha Genome - miRNA gene...

miRNAs and Cancer

Sponsored by:

Participating Experts:George Calin, M.D./Ph.D.

The University of TexasM.D. Anderson Cancer CenterHouston, Texas

Frank Slack, Ph.D.Department of Molecular, Cellular and

Developmental BiologyYale UniversityNew Haven, CT

Scott Hammond, Ph.D.University North Carolina at Chapel Hill

Chapel Hill, NC

Brought to you by the AAAS/Science Business Office

Webinar SeriesScience

20 February, 2008

A non-codingRNA revolution in the

cancer society

George Adrian Calin, MD, PhD

Associate Professor

Experimental Therapeutics Dept

Univ. Texas, MD Anderson CC

Houston, US

Year 2K “Central dogma” of molecular oncology

Cancer is the GENETIC DISEASE with the most complex mechanism.

Oncogenes and Tumor-suppressors are the two types of PROTEINS deregulated in cancer

cells.

Normal cell

PROTO-ONCOGENES

TUMOR

SUPPRESSORS

Malignant cell

ONCOGENEStumor suppressors

microRNA

I am very smalland non-coding!But I can do a big jobin your cells!Size doesn’t matter!

Really?! Who are you little thing? The son of a new genetics?

messengerRNAs

Cytoplasm

ORF

mRNA cleavage

RISC RISC

RISCTranslation repression

Some miRNAs

Pre-miRNA(hairpin precursor)

Drosha

Genome - miRNA gene

Pri-miRNA

Duplex

Dicer

miRNA

NucleusExportin-5

RISC

3’UTR

Small RNAs make big splash

Lee RC, Feinbaum RL, Ambros V. Cell. 199 3 Dec 3;75( 5):843. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinge r JC, Rougvie AE, Horvitz HR, Ruvkun G. Nature. 2000 Feb 24;403(6772 ):901.

How many miRNAs are enough for what?

~15,000 conserved human stem loops,

many more non-conserved, but potentially important

(Bentwich et al, Nat Genet, 2005; Berezikov et al, Cell 2005; Cummins et al, PNAS, 2006)

ALTERATIONS OF MICRORNAS ARE FOUND IN EVERY

TYPE OF HUMAN CANCER

miRNA

expression profiles

classify

human leukemias

and

carcinomas

(Calin et al, PNAS 2002; Lu et al, Nature, 2005; Volinia & Calin et al, PNAS 2006; Landgraf et al, Cell 2007)

MNC Ly CD5

Cluster 2 Cluster 1

CLL

A

246

109

544151

63

138

6618 50

LUNG carcinoma

BREAST carcinoma

COLON carcinoma

GASTRIC carcinoma

Endocrine PANCREATIC tumors

PROSTATE cancers

B-CLL

papillary THYROID carcinoma

primary GLIOBLASTOMAS

HEPATOCELLULAR carcinoma

B

836

(Calin et al, PNAS, 2004; Esquela-Kerscher & Slack, Nat Rev Cancer 2006; Calin & Croce, Nat Rev Cancer 2006)

miR Amplification

miR Translocation

miR Mutation

miR Deletion/

Hypermethylation/

Abmormal processing

AAAA

AAAAAAAA

AAAA

AAAA

AAAAAAAA

AAAA

PCG Amplification

PCG Translocation

AAAA

AAAA

PCG Mutation

PCG Deletion/Hypermethylation

AAAA

AAAA

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

Oncogenic proteins

NH2COOH

NH2COOH

Tumor-suppressor proteins

Low apoptosis

High proliferation

Metastasis

High angiogenesis

NH2COOH

Causing miRNA abnormal expression - the combinatorial “in cascade”model

(Calin & Croce, Cancer Res, 2006; Calin & Croce, J Clinical Inv 2007)

MICRORNAS AS ONCOGENES AND TUMOR

SUPPRESSORS

miRNAs as

tumor sup pressors

(e.g.miR-16/15a, let-7) or

oncogenes

(e.g. miR-155, miR-21,

miR17-92 cluster,

miR-10b)

(Johnson et al, Cell 2005; He et al, Nature 2005; O’Donnell et al, Nature 2005;

Cimmino et al, PNAS 2005; He et al, Nature 2007; Chang et al, Mol Cell 2007;

Raver-Shapira et al, Mol Cell 2007; Ma et al, Nature 2007;

Fabbri et al, PNAS 2007; Chang et al, Nat Genet 2008; Tavazoie et al,Nature 2008)

Akt-PKB

Proliferation

Apoptosis

PDCD4

c-JUN

miR-21

PTEN

PI3K

miR-17-92 E2F1 c-MYC

MAPK pathway RAS let-7

ERK5 miR-143/145

TCL1miR-29b

miR-181b

ARFTP53

TP53INP1

miR-155

BCL6

miR-127

miR-15a/16-1

BCL2

TSGs

DNMT3A/3B

miR-29a, -29b, -29c

miR-34

ALTERATIONS OF MICRORNAS AND/OR TARGET

INTERACTION MAY CAUSE CANCER PREDISPOSITION

Mutations

in miRNAs and

“interactor” SNPs

as possible

predisposing

factors in human

cancers

Germline abnormalities in miR15a/miR16-1 transcript are associated with

CLL and breast cancer aggregation

mir-16-1 normal genome

precursor

mir-16-1 (CtoT)+7

+1 +7precursor

A

U6

A

P

miR

16

WT

Em

pty

V

29

3

miR

16

-1

MU

T

miR

16

WT

Em

pty

V

29

3

miR

16

-1

MU

T

GFP

miR-16-1 miR-15a

C

0

5

10

15

20

25

30

35

Mutant 15-16 Empty

vector

Non

transfected

Ct

va

lue

s

1ng RNA

5ng RNA

10ng RNA

miR-16-1

MEG01 cells

DB

NB Ratio 0.25 0.50 2.07 1.22

MAr Ratio 1.01 3.25 9.20 6.24

miR-16-1

NB Ratio 0.29 0.51 1.97 1.37

MAr Ratio 0.95 1.30 3.50 2.30

miR-15a

U6

(Calin et al, N Engl J Med, 2005; Raveche et al, Blood 2007)

SNPs in interactor sites could predispose to familial breast cancer by

affecting the microRNA targeting

(Nicoloso & Sun et al, in preparation, 2008)

CC

/TT

0.7

0.8

0.9

1

1.1

1.2

miR-101 scrambled

TP53BP2_miR-101

T/C

0.8

0.9

1

1.1

1.2

1.3

miR-638 scrambled

BRCA1

AAGUC

AAUAgUG uCAUGACAU

TAATtAC gGTACTGTA

ACATC T

miR-101TP53BP2_T

-16.82 151

miR-101TP53BP2_C

-11.41 123

AAGUC A A

A UAgUG uCAUG CAU

T ATtAC gGTAC GTA

ACATC A T C

p 0.0039

C A

UC CGG CgGUgggCG GgC G UAgGG

AG GCC GtCAtttGC CtG C AAgCC

C A T TTTT A A

-25.53 94

miR-638BRCA1_T

C U A

UC CGG CgGUgggCGgG GC AggG

AG GCC GtCAtttGCtC CG TttC

C A T A

-27.52 114

miR-638BRCA1_C

p 0.0163

miR binding site containingTARGET SNP

Promoterluciferase

disrupting allele

HIGH

Promoter

luciferaseactivating allele

Low

LUCIFERASE ACTIVITY

pGL3-SNP ALLELIC

PAIRS

disrupting allele

activating allele

Expected

> 1RATIO of pGL3-SNP

LUC ACTIVITY

disrupting allele

activating allele

RA

TIO

C/T

RA

TIO

T/C

pGL3-TP53BP2 C/T pGL3-BRCA1 T/C

MIRNA PROFILING AS A NEW DIAGNOSTIC & PROGNOSTIC

TOOL FOR CANCER PATIENTS

miRNAs expression

signatures associated

with diagnosis and

prognostic factors

(CLL, DLBCL, Lung,

Colon, Pancreas,

Brain ca.)

(Michael et al, Molec Cancer Res 2003; Lu et al, Nature, 2005; Eis et al, PNAS, 2005

Lui et al, Cancer Res 2007, Bloomston et al, JAMA 2007; Mi et al, PNAS, 2007; Garzon et al, Blood in press 2008)

A unique miRNA signature is associated with

lung cancer prognosis

Table 5.

(Yanaihara et al, Cancer Cell, 2006)

A unique miRNA signature is associated with CLL prognosis

Nr. Crt.

Component Map P value

Aggressive CLL **

Observation***

1 miR-15a 13q14.3 0,018 high cluster 15a/16-1 del CLL & Prostate ca.

2 miR-195 17p13 0,017 high del HCC

3 miR-221 Xp11.3 0,010 high cluster 221/222

4 miR-23b 9q22.1 0,009 high cluster 24-1/23b FRA 9D; del Urothelial ca.

5 miR-155 21q21 0,009 high amp child Burkitt’s lymphoma 6 miR-223 Xq12-

13.3

0,007 low normally expression restricted to myeloid lineage

7 miR-29a-2 7q32 0,004 low cluster 29a-2/29b-1 FRA7H; del Prostate ca.

8 miR-24-1 9q22.1 0,003 high cluster 24-1/23b FRA 9D; del Urothelial ca.

9 miR-29b-2 1q32.2-32.3

0,0007 low

10 miR-146 5q34 0,0007 high

11 miR-16-1 13q14.3 0,0004 high cluster 15a/16-1 del CLL, prostate ca.

12 miR-16-2 3q26.1 0,0003 high identical miR-16-1

13 miR-29c 1q32.2-32.3

0,0002 low

Note: * - All the members of the signature are mature microRNAs; ** - Aggressive CLL is represented by group 1 includes patients with IgVh unmutated and Zap-70 positive (high), both predictors of poor prognosis. *** - FRA = fragile site; del = deletion; HCC = hepatocellular carcinoma; ca. = carcinoma.

(Calin et al, N Engl J Med, 2005)

MICRORNAS AS NEW THERAPEUTIC TARGETS &

NEW DRUGS

miRNAs as cancer drugs

miRNAs as targets

for cancer drugs

(Krutzfeldt et al, Nature 2005; Weidhaas et al, Cancer Res 2007)

Principles of

microRNA-based

Gene Therapy

MIRNA EXPRESSION

IN NORMAL CELL

(Tili et al, Future Oncology, 2007)

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

NH2COOH

miR-16 & miR-15 based Gene Therapy in CLL

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

7 15 21

Days

Tum

or V

olu

me (

mm

3)

Mock pRS-E pRS15/16A MEG01

MOCKMEG01pRS-E

MEG01pR15/16

B

C

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Mock pRS-E pRS15/16

Day 28

(Calin, Cimmino & Fabbri et al, PNAS in press, 2008)

Opening Pandora’s box:

Revisiting the molecular oncology dogma

Mad spirit

of cancer

proteins

miRNAs

supressorsmiRNAs

activators

protein

s

megaRNAs

Ultraconserved

Genes

UCG expression profiles classify human cancers

* 50 UCRs differentiate

human cancers

(P<0.005)

* half of them Non-exonic

* 6/12 Exonic UCRs represent

ANTISENSE transcripts with

host gene

CLL

CRC

HCC

133 cancer samples

(Calin & Liu & Ferracin et al, Cancer Cell, 2007)

MicroRNAs and genomic dark matter alterations in human leukemias

and carcinomas

CCC-OSU

Croce’s laboratory

Carlo M. Croce

Amelia Cimmino,

Sylwia Wojcik,

Muller Fabri,

Ramiro Garzon,

Arriana Bottoni,

Gianpiero di Leva,

Esmerina Tili,

Jean-Jacques Michaille,

Cristian Taccioli,

Hansjuerg Alder,

Stefano Volinia,

Chang-gong Liu,

A. de la Chapelle’s lab

Huiling He,

Krystian Rajewsky

Univ of Ferrara

Negrini’s lab

Massimo Negrini,

Manuela Ferracin.

OSU-Pharmacology

Tom Schmittgen,

Eun Joo

TJU-KCC

Philadelphia

Cinzia Sevignani,

Terry Hyslop,

Linda Siracusa

Romania

Dragos Stefanescu,

Vlad Herlea

(Fundeni Hospital)

Natl Inst Cancer, Genoa

Simona Zuppo

MD Anderson CC

Calin’s laboratory

(CLL Global Research Found. &

MD Anderson Trust &

Regents Research Scholar )

Milena S. Nicoloso, MD

Riccardo Spizzo, MD

Masayoshi Shimizu, BS

Simona Rossi, PhD

Sumaiyah Rehman, PhD student

CLL research

Thomas J. Kipps

Michael Keating

Kanti Ray

Neil Kay

Susan Slager

Laura Rassenti

Donna Neuberg

John Byrd

How can we find targets?I am getting crazy to find them!

It is very difficult…we could do…cfyyou… gvcjgyf? Buywe? hegruey?!

534uiytfdu3..12:1247 = fybrbc + fxnvhr jhwhuerh .34@ = fshxbf

LKM / KUHF..23649 % 7(346) IWRE CIRE

I am not sure that is going to work and it will take forever?!

Maybe we could askin the microarray facility to customize microchips for miRNA genes!

ihsa-miR-143

ugagaugaagc

acuguagcuca

Cinzia Sevignani 2005

Enjoy the ncRNA revolution!

miRNAs and Cancer

Sponsored by:






Chapel Hill, NC



20 February, 2008

MicroRNAs and cancer

Frank Slack

[email protected]

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Thousands of microRNAs act in multiple

biological events

Aging

Metabolism

Developmental

timing

Apoptosis

Cell fate/differentiation, Cell cycle…

Differentiation

…Cancer

MicroRNAs were discovered by V. Ambros

and G. Ruvkun in C. elegans

cuticle

mouth

Lee et al. 1993 Cell

Reinhart et al. 2000 Nature

Calin, G.A., et al., 2004. PNAS 101:2999-3004

microRNA genes map to cancer loci

MicroRNAs

are mis-

expressed in

cancer

Down-regulated in colon

Down-regulated in lung

Down-regulated in thyroid

Down-regulated in all

Down-regulated in breast

Up-regulated in all

Up-regulated in allD. Brown, 2002

Calin et al. (2004) Proc. Natl. Acad. Sci. USA 101, 2999-3004

Oncomirs - miRNAs and cancer

Oncogene miRNAsmir-17-92; mir-155, mir-21

Tumor suppressor genesLATS; RB1; PTEN

Tumor suppressor miRNAsmir-15/16; let-7; mir-34 Oncogenes

RAS; MYC

BCL2; MET

TSG

ONCOGENE

Oncogenic microRNA

TSG

ONCOGENE

Oncogenic microRNA

Normal cell Tumor cell

Normal cell Tumor cell

TS microRNA TS microRNA

TSG

OncoProtein

MicroRNAs act as oncogenes and tumor suppressors in

cancer

MicroRNAs commonly associated

with human cancerTable 1. MicroRNAs associated with human cancers

miRNA Gene Loci Cancer association Function* References

miR15,

miR-16

chromosome 13q14

Frequently deleted/downregulated in B-cell chronic

lymphocytic leukemia.

Negatively regulates the anti-apoptotic gene, BCL2.

TS

Calin, 2002

Cimmino, 2005

miR-143,

miR-145

chromosome 5q32-33

Decreased abundance in colorectal cancer.

Down-regulated in breast, prostate, cervical, and

lymphoid cancer cell lines.

miR-145 decreased in breast cancer.

TS

Michael, 2003

Iorio, 2005

miR-21

chromosome 17q23.2

Antiapoptotic factor.

Upregulated in glioblastomas and breast cancer.

OG

Chan, 2005

Ciafre, 2005

Iorio, 2005

let-7

multiple loci

Negatively regulates the Ras oncogene.

Directs cell proliferation, differentiation.

Decreased abundance in lung cancer.

TS

Johnson, 2005

Takamizawa, 2004

miR-142

chromosome 17q22

t(8,17) translocation that places the MYC oncogene

downstream of the mir-142 hairpin resulting

in an aggressive B cell leukemia due to

MYC over-expression.

N/A

Lagos-Quintana, 2002

BIC/miR-155

chromosome 21q21

Upregulated in pediatric Burkitt’s lymphomas,

Hodgkins, primary mediastinal and

diffuse large B cell lymphomas.

Upregulated in human breast cancer.

OG

Eis, 2005

Kluiver, 2005

van den Berg, 2003

Metzler, 2003

Iorio, 2005

miR-17-19b

cluster

chromosome 13q31-32

Upregulated by the c-Myc oncogene.

Negatively modulates E2F1 oncogene.

Loss-of-heterozygosity of cluster in

hepatocellular carcinoma.

Over-expressed in B-cell lymphomas.

TS/

OG

He, 2005

O’Donnell, 2005

*Abbreviations: TS, tumor-suppressor gene; OG, oncogene; N/A, not applicable

MiRNAs and cancer- a summary

• miRNAs control cell cycle, cell differentiation and

apoptosis by regulating oncogenes and TS genes

• miRNAs are misexpressed in cancer and are therefore

excellent diagnostic/prognostic markers in cancer

• Some miRNAs e.g. mir-155, can cause cancer and

oncogenic miRNAs may be therapeutic targets in

cancer

• Other miRNAs like let-7, may prevent cancer and may

be therapeutic molecules themselves.

• MicroRNAs could augment current cancer therapies.

Thanks to all members of the Slack LabCurrent Post-docs:Shih-Peng ChanAlex deLencastre Masaomi KatoPedro Medina Ryusuke NiwaZach PincusGiovanni StefaniPhong Trang

Current Grad students:Imran BabarKatherine Carter Lena Chin Sirie Godshalk Sarah Roush Mike Turner

Technician:Xianping Liang

Collaborators:Joanne Weidhaas at Yale UniversityDavid Brown/Lance Ford at Ambion/Asuragen

Former Post-docs:E-Y. Choi, Helge Grosshans, Gopi Ramaswamy,Diya Banerjee, Aurora Kerscher

Former Grad students:Steven Johnson, Monica VellaBetsy Schulman, Mona NoldeMichelle BoehmFormer Technicians:Shin-Yi Lin, Kristy Reinert

Thanks to:NIH, NSF, Patterson Trust, YCCMSD, Rockefeller Fund, Asuragen, CT Dept of Public Health, Ellison Medical Foundation, AFAR, McDonnell Foundation

miRNAs and Cancer

Sponsored by:






Chapel Hill, NC



20 February, 2008

miRNAs and Cancer

Scott Hammond, Ph.D.

University North Carolina at Chapel Hill

Chapel Hill, NC

Do microRNAs play a causal role in human cancer?

Identification of microRNA genes

Analysis of microRNA expression in cancer


How do microRNAs promote/suppress cancer?

Regulation of cell proliferation

Regulation of apoptosis

Propagation of tumor stem cells

miR p27

miR Bax








miR p27

miR Bax



Can microRNAs be targeted for therapy?

CAPAAAAAAAAAAAAAAAA

TTTTTTTTTTTTTTT

Most microRNAs were discovered by cloning

mRNA

CAPAAAAAAAAAAAAAAAA

P OH

TTTTTTTTTTTTTTT


mRNA

microRNA

CAPAAAAAAAAAAAAAAAA

P OH

TTTTTTTTTTTTTTT

P OHP OH


mRNA

microRNA

PP OH AAAAAAAAAAAAAAAA

TTTTTTTTTTTTTTT

541 microRNAs have been identified in the human genome

Taken from: Mariana Lagos-Quintana, Reinhard Rauhut, Winfried Lendeckel, Thomas Tuschl, Science, 294: 853-858 (2001)

MicroRNA expression analysis using microarrays

Thomson et al, Nature Methods 1(1): 47-53 (2004)

MicroRNA expression during

Mouse development

A polycistronic cluster of microRNAs are overexpressed in cancer

Ch13-ORF25

Proliferation

Apoptosis

Angiogenesis

Invasiveness

OncomiR-1 (Oncogenic microRNA-1)

OncomiR-1 expression accelerates lymphoma development

He et al, Nature 435(7043): 828-833 (2005)

Matsubara H, et al. Apoptosis induction by antisense oligonucleotides against miR-17-5p

and miR-20a in lung cancers overexpressing miR-17-92. Oncogene. 2007 Mar 26;

[Epub ahead of print]

Wang CL, et al. Activation of an oncogenic microRNA cistron by provirus integration.

Proc Natl Acad Sci U S A. 2006 103(49):18680-4.

Volinia S, et al. A microRNA expression signature of human solid tumors defines cancer

gene targets. Proc Natl Acad Sci U S A. 2006 103(7):2257-61.

Hayashita Y, et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in

human lung cancers and enhances cell proliferation. Cancer Res. 2005 65(21):9628-32.

O’Donnell KA, et al. c-Myc-regulated microRNAs modulate E2F1 expression. Nature.

2005 435(7043):839-43.

Dews et al. Augmentation of tumor angiogenesis by a Myc regulated microRNA

Nature Genetics 38(9): 1060-1065 (2006)






miR p27

miR Bax



Can microRNAs be targeted for therapy?

MicroRNAs as therapeutic targets

AAAAAAAAAAAA

oncomiR containing RISC

Translational arrest

(Oncogenic microRNA)


AAAAAAAAAAAA

oncomiR containing RISC

Translational re-activation

(Oncogenic microRNA)

AntagomiR


AAAAAAAAAAAA

Insufficient TS-miR RISC

Translational activation

(Tumor suppressor microRNA)


AAAAAAAAAAAA

Translational arrest

(Tumor suppressor microRNA)

TS-miR containing RISC

miR mimic

The microRNA hypothesis of cancer

Tumors downregulate many microRNAs to promote

a “stem cell” like state

Selected microRNAs (oncomiRs) are expressed at a

high level to promote proliferation, suppress apoptosis

What is the mechanism for widespread alterations in gene

expression? Transcriptional, processing, turnover?

Drosha processing of “tumor suppressor” miRNAs

is inhibited in early development (and cancer?)

Acknowledgements

Chuck Perou

Joel Parker

Gerry Usary

Jason Leib

Cheol-koo Lee

Joel Parker

Tricia Wright

Elizabeth Morin

James Tsuruta

Debra O-Brien

Yi Zhang

Dazhi Wang

Hammond lab members:

Mike Thomson

Summer Goodson

Keith Woods

Marty Newman

Handan Kaygun

Andrew Baraniak

Vidya Mani

Former lab members:

Jerry Bernards

Emily Kuty

UNC Microarray Facility

Software from Michael Eisen

Department of Cell and Developmental Biology

General Motors Cancer Research Foundation

NIH-NIGMS

American Cancer Society

American Association for Cancer Research

Greg Hannon

Lin He

Jidong Lu

Michelle Carmell

David Mu

Scott Powers

Carlos Cordon-Cardo

Scott Lowe

Mike Hemann

Yue Xiong

Antonio Giraldez

Paul Barnes

Tom Maynard

Look out for more webinars in the series at:

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miRNAs and CancerWebinar SeriesScience

20 February, 2008


For related information on this webinar topic, go to: www.stratagene.com/miRNA

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Webinar Series miRNAs and Cancer · Pre-miRNA (hairpin precursor) Drosha Genome - miRNA gene...

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