mi rnasexpression in oesophagealand gastriccancers · cech tr et al -pnas 1979 …ribosomal...

Matteo FASSANUNIVERSITÀ DEGLI STUDI DI PADOVA, ITALIA

Hot Issues in Molecular PathologyPopper H, Schaff Z, Tímár J

Technology Transfer in Diagnostic Pathology

7th Central European Regional Meeting

Siófok - Hungary, May 14-16 , 2012

MIRNAS expressionin oesophageal and gastric cancers

MIRNAS expressionin oesophageal and gastric cancers

MULTISTEP MODELOF SCIENTIFIC PARADIGMS

1761 1858 1953

Die ZellularpathologieDe sedibus et causis

morborum per anatomen indagatis

Molecular structure of nucleic acids; a structure

for deoxyribose nucleic acid

THE CODING-RNA PARADIGM

DNA

Gen

e

AA

CG

GC

TG

CG

CA

DNA strand

UUCC

GGCC

GGGG

GGUU

AACC

CCUU

mRNA

TRANSCRIPTION

protein

Cod

onTRANSLATION

The begin of a new story…

Holley RW et al - Science 1965

…the first non-coding RNA to be characterised was the alanine tRNA found in baker's yeast…

Cech TR et al - PNAS 1979

…ribosomal ribonucleic acid (rRNA) is the RNA component of the ribosome, the protein manufacturing machinery of all living cells…

NON-CODING RNAS’ WORLD

Bartel DP - Cell 2004

Small RNAS’ WORLD

THE INTERFERENCE ERA

Fire A et al - Nature 1998

…reported a potent gene silencing effect after injecting double stranded RNA into C. elegans…

ONCOMIRS: the new actors in the cancer scenario

Visone R & Croce CM - Am J Pathol 2009

Baffa R, Fassan M & Croce CM - Laborwelt 2009

Fassan M, Rugge M & Baffa R - Front Biosci 2011

… one-third of protein-coding human mRNAs are susceptible to this complex miRNA regulatory network…

let-7

RAS; C-MYC

Normal Cancer

miRNA as tumor suppressor gene

miR-21

PDCD4

miRNA as oncogene

PNASHuman microRNA genes are frequently located at fragile sites and genomic regions involved in cancers

Calin GA et al - PNAS 2003 Volinia S et al - PNAS 2005

A microRNA expression signature of human solid tumors defines cancer gene targets

Iorio MV & Croce CM – J Clin Oncol 2009

Mechanisms of miRNAs’ dysregulation in cancer

Baffa R, Fassan M, Gardiman M, Rugge M, Croce CM - J Pathol 2009

Fassan M, Palazzo JP, Rugge M, Croce CM - Breast Cancer Res 2009

The use ofFF-PE tissues

The pathologist’s point of view

PDCD4 nuclear loss inversely correlates with miR-21 levels in colon carcinogenesis

Fassan M, Pizzi M, Mescoli C, Ludwig K, Rugge M - Virchows Arch 2011

Fassan M, Pizzi M, Balistreri M, Rugge M - Dis Esophagus 2011

Programmed cell death 4 nuclear loss and miR-21 or activated Aktoverexpression in esophageal squamous cell carcinogenesis.

PRENEOPLASTIC LESIONS




The direct visualization in FF-PE tissues - ISH

MIRNAS IN SITU HYBRIDIZATION

Van Baal J, Fassan M, Rugge M, Siersema P – Gut 2012




The identification of the target genes - IHC

+3.00

0.00

-3.00

hsa-miR-205

N IM LG HG BAchsa-let-7c

hsa-mIR-605hsa-miR-100

hsa-miR-203

hsa-miR-99ahsa-miR-23ahsa-miR-147hsa-miR-192hsa-miR-215hsa-miR-560hsa-miR-326hsa-miR-615

Up

Down

13 MIRNAS DIFFERENTLY EXPRESSED IN BE-ONCOGENESIS:

hsa-miR-147 hsa-miR-192 hsa-miR-215 hsa-miR-326 hsa-miR-560 hsa-miR-615-3p

6 UP-REGULATED hsa-let-7chsa-miR-23a hsa-miR-99a hsa-miR-100 hsa-miR-203 hsa-miR-205 hsa-miR-605

7 DOWN-REGULATED

Fassan M, Croce CM, Rugge M – Int J Cancer 2011

hsa-let-7c TARGETS HMGA2/HMGI-C

� HMGA2/HMGI-C is a direct target of hsa-let-7c;

� HMGA2/HMGI-C is a small non-histone chromosomalprotein that modulates transcription;

� HMGA2/HMGI-C over-expression is a hallmark of variousbenign & malignant human tumors, including Gastric Cancer.

HMGA2/HMGI-C IHCRing chromosome

Differential Diagnosis: Liposarcoma lipoma-like versus lipoma.

N

20%

60%

100%

N IM LG HG BAc

BARRETT’S ONCOGENESIS: HMGA2 IHC

BAcHG

N

BAc

� Biopsy samples:

N=25, IM=25, LG=16, HG=11, BAc=10

� Score Values: 0= 0-5%; 1= 6-50%; 2= >50%

p < 0.001

Fassan M, Croce CM, Rugge M – Int J Cancer 2011

Fassan M, Croce CM & Rugge M – WJG 2011

The clinician’s point of view

miRNAs as new prognostic tools

1.0

1.0

0.5

Su

rviv

al p

rob

abil

ity

Time

1.0

0.5

Surv

ival

pro

babi

lity

Time

Kogo R et al – Clin Cancer Res 2011

…the miR-146a expression status is associated with patients’survival and the presence of lymph node metastases; miR-146ais an independent prognostic factor in GC…

high miR-146a

low miR-146a

MIR-27a POLYMORPHISM & GASTRIC ATROPHY

Arisawa T et al – Dig Dis Sci 2007

…the miR-27a genome region polymorphism may be an important definitive factor to develop the gastric mucosal atrophy in Japanese…

100

0

50

% o

f gas

tric

atr

oph

y

A/A G/G A/G



miRNAs as non-invasive diagnostic

markers

MIRNAS AS PLASMA BIOMARKERS IN GC

Tsujiura M et al – BJC 2010

Plasma miRNAs are dysregulated in gastric cancer patients

7.0

3.0

1.0

5.0

.

.

.

.Rel

ativ

e qu

anti

ty

Normal Cancer

7.0

3.0

1.0

5.0

.

.

.

.Rel

ativ

e qu

anti

ty

Beforesurgery

Aftersurgery

miR-21 miR-21

Plasma miRNAs expression after 1 month from surgery



miRNAs as biomarkers of therapeutic response

MIRNAS AND MULTI-DRUG RESISTANCE

Xia L et al – Int J Cancer 2008

miR-15b and miR-16 modulate MDR by targeting BCL2 in human gastric cancer cells

1

0

0.5

bcl2

rel

ativ

e ex

pre

ssio

n

miR15b miR16

1

0

0.5

bcl2

rel

ativ

e ex

pre

ssio

n

miR15b miR16

miR15b miR16ctrl

bcl2

β-actin

40

0

20A

pop

tosi

s %

3000

0

1500

Cas

pas

eac

tivi

ty

microarray qRT-PCR WB



miRNAs as new therapeutic tools

MicroRNA Replacement Therapy for miR-145 and miR-33aIs Efficacious in a Model of Colon Carcinoma

polyethylenimine (PEI)-mediated delivery of unmodified miRNAs

miR-145 delivery reduced tumor proliferation and increased apoptosis, with concomitant repression of c-Myc and ERK5

Ibrahim AF et al – Cancer Res 2011

Beyond miRNAs…the UCRs(ultraconserved regions)

� noncoding RNA (ncRNA) transcripts (longer than 200 bp)

� a subset (n=481) of conserved sequences that are located in both intra- and intergenic regions

� conserved (100%) between orthologous regions of the human, rat, and mouse genomes

Fassan M & Rugge M – Unpublished data

2.0

0

BM

vs

N

uc.153 uc.472 uc.473

1.0

Human

2.0

0

1.0

BM

vs

N

uc.153 uc.472 uc.473

Mouse (model)2.0

0

BM

vs

N

uc.153 uc.472 uc.473

1.0

Rat (model)

Gastroduodenal anastomosis

CONCLUSIONS (1/2)

miRNAs expression patterns have a significant impact on human disease

miRNAs are dysregulated in GI diseases: from inflammation to cancer

miRNAs are fundamental in all the cellular processes

CONCLUSIONS (2/2)

miRNA expression profiling identifies cancer gene targets

Regulating miRNA expression might be a novel strategy for gastrointestinal tumors chemoprevention

miRNAs as novel diagnostic and prognostic tool

Laboratory of surgical pathology - Surgical pathology & cytopathology unit – University of Padua

EBRA registry (Barrett’s esophagus Veneto region registry; www.esofagodibarrett.org)

Ohio State University - Comprehensive Cancer Center

Istituto Oncologico del Veneto - Surgery & endoscopy units

Funding agencies

…a particular thank to…

Laboratory of molecular pathology - Surgical pathology & cytopathology unit - University of Padua

mi rnasexpression in oesophagealand gastriccancers · cech tr et al -pnas 1979 …ribosomal...

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