2009 June MGEL W/S

성 현 아

MicroRNA & Carcinogenesis

2009 June MGEL W/S성 현 아

Presentation Summary

• MicroRNA, Discovery and Biogenesis

• MicroRNA & Carcinogenesis

• MiRNA and Breast Cancer

How few genes there were !There weren't that many more than a fruit fly or

a unicellular organism, yeast.

Saccharomyces cerevisiae~ 6,000 genes

Drosophila melanogaster~ 14,000 genes

Homo sapisens~ 19,000 genes

24 March 2000 3 April 200129 May 1997

What makes human to be human?

"Junk" RNA May Have Played Role in Vertebrate Evolution

Right about when vertebrates split off fro

m other animals, there was an explosion o

f these microRNAs in their DNA.

The complexity didn't come from the numb

er of genes an animal has.

The complexity comes from how similar g

enes are used.

This is where microRNAs come in because

they affect how an animal (or plant) uses it

s genes.

MicroRNAs Control How Genes are Used.

Evidence that Vertebrate Complexity Comes from non-codingRNA

Non coding RNA

• A non-coding RNA (ncRNA) is any RNA molecule that is not translated into a protein

• tRNA / rRNA

• snRNA/miRNA/gRNA/piRNA/siRNA/tmRNA

• small RNA(sRNA), non-messenger RNA (nmRNA), small non-messenger RNA (snmRNA), or functional RNA (fRNA)

• lin-4 is essential for the normal temporal control of diverse postembryonic developmental events in C. elegans

• lin-4 downregulates LIN-14

larval stage (L1)

• lin-4 does not encode a protein

• Two small lin-4 transcripts of approximately 22 and 61 ntsequences complementary to a repeated sequence element in the 3′ UTR of lin-14 mRNA

• lin-4 regulates lin-14translation via an antisense RNA-RNA interaction.

The Biogenesis of miRNA & Assembly into miRISC

Txn. Of pri-miRNA(RNA pol II) pri-miRNAs pre-miRNA(Drosha)

Transport into cytoplasm small RNA duplexes(Dicer)

miRISC(RNA-induced silencing complex) target mRNA sequence

target miRNA silencing

Possible Mechanisms of miRISC-Mediated Repression : post-transcriptional gene regulation

Usually via either translational repression or direct mRNA cleavage

MicroRNA(miRNA)

• Endogenous 22-nt non-coding RNAs (22-24nts)

• Protein level regulation in a sequence-specific manner

• Developmental timing in worms, cell death and fat metabolism in flies,

and leaf development and floral patterning in plants(Evolutionary conserved)

• In mammals, including cell growth and apoptosis, hematopoietic lineage differentiation, insulin secretion, brain morphogenesis, and muscle cell differentiation and proliferation

• From 450 to 1000 miRNAs in the human genome

• Most human protein-coding genes influenced

• A single miRNA may bind to as many as 200 target genes

• Half of the known miRNA mapped to fragile sites in the genome, providing the first clue to their role in cancer

At least 10% of the protein coding mRNAs might be conserved targets of miRNA

Expression and function of micro RNAs in immune cells during normal or disease state Esmerina Tili1, Jean-Jacques Michaille1, 2, George Adrian Calin 3

Int J Med Sci 2008; 5:73-79

microRNA and Human Disease

Potential mechanisms of aberrant miRNA regulation

Mutation/SNP in the 3’ UTR target mRNA

(poly-miRTS)miRNAs located near fragile sites

epigenetic changes Roughly 20,000 poly-miRTS catalogued in databases such

as PolymiRTS(http://compbio.utmem.edu/miRSNP/)

Method to determine miRNA expression levels

• High-throughput tech. for hundreds of miRNAs in a large number of samples(commonly used )

• Bead-based flow-cytometric tech.

• miRAGE : a genome-wide miRNAanalysis with serial analysis of gene expression (SAGE)

• qRT PCR

miRNA & Carcinogenesis

How miRNA contribute to oncogenesis ?

1> Function either as tumor suppressors or oncogenes

2> The genomic abnormalities found to influence the activity of miRNA : frequently located near fragile sites

3> Epigenetic changes such as alterations in CpG methylation patterns

4> SNP and/or mutations in the miRNA themselves

5> Differences in the expression levels or affinities of any protein involved in miRNA Transcription and Processing

: Drosha, Dicer, Argonaut etc.

6> the role of polymorphisms in the sites of target mRNAs 3’ UTR

miRNAs as cancer players: miRNA , frequently located near fragile sites

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

© 2004 by National Academy of Sciences

A model of miRNA involvement in cancer by modulation of expression of tumor suppressor genes and oncogenes

Carlos Caldas et al. Sizing up miRNAs as cancer genes, Nature Medicine 11, 712 – 714 (2005)

miRNA-expression profiles classify human cancers.

• 217 miRNA, 334 samples including multiple human cancers

• A general downregulation of miRNAs in tumours compared with normal tissues

• Classifying poorly differentiated tumours using miRNA expression profiles

• A. Most of miRNA (129 out of 217, P<0.05) had lower expression levels in human tumour samples

• B. Normal lungs and lung adenocarcinomas of K-RasLA1 mice

• C. HL-60 cells, treated with all-trans retinoic acid or vehicle

• Highlightening the potential of miRNA profiling in cancer diagnosis

• miR15 and miR16 are deleted or down-regulated in the majority

(≈68%) of CLL cases.

• miR15 and miR16 are located at chromosome 13q14, a region

deleted in more than half of B-CLL

• Genome wide expression profiling of miRNAs in human B-CLL

using microarray

• 41 samples from 38 pts. with CLL and 6 normal samples

( 1 lymph node, 2 tonsillar CD5+ B cells, 3 blood MNC)

Top 25 miRNAs differentially expressed in CLL cells vs. CD5+ cells

Calin G. A. et.al. PNAS 2004;101:11755-11760

miRNAs differentially expressed bet. the two main CLL clusters

miRNA signatures associaed with prognosis in B-CLL

• 1st Set – 94 pts. of CLL cells

2nd Set - 50 pts. of CLL cells

to validate the predictive

power of the miRNA signature

• Northern blotting

miRNA-microchip

procedures( 368 probes,

corresponding to 245 human

and mouse miRNA genes)

Prognostic Factor

ZAP-70 >20% ZAP-70 <20%

IgVh status unmutated

Group 1(36pts) Group3(10pts)

IgVh status mutated

Group 2(10pts) Group 4(47pts)

• Group1

Poor prognosis

(unmutated IgVh& high ZAP-20)

• 13 miRNAassociated with prognostic factors

Relationship bet. the level of expression of miRNA and the time from diagnosis to initial therapy

• 94 pts

• 9 miRNAs, all members of the 13-member prognostic signature

• Short interval(40±39 months)/ longer interval (88±42 months)

• miRNA expression can be included in the markers with prognostic significance in CLL

• Germ-line or somatic mutations were found in 5 of 42 sequenced miRNAsin 11 of 75 pts. with CLL, but no such mutations were found in 160 subjects without cancer

tool.

• 104 pairs of primary lung cancer and corresponding normal tissue

• 43 miRNAs differentially expressed in lung cancer vs. normal tissue thru microarray

• 6 miRNAs differentially expressed in adenocarcinoma and squamous cell carcinoma

• Correlation bet. miRNA expression profiles and prognosis of lung adenocarcinoma pts

• Validation using an independent set of adenocarcinoma

• Using real-time RT-PCR, miRNA expression profiling in 112 NSCLC pts. divided into the training and testing sets

• a five-miRNA signature for the prediction of treatment outcome of NSCLC in the training set

• Validation by the testing set and an independent cohort

• Patients with high-risk scores in their miRNA signatures had poor overall and disease-free survivals compared to the low-risk-score pts.

This miRNA signature is an independent predictor of the

cancer relapse and survival of NSCLC patients.

2008 Jan08

Calin and Croce Nature Reviews Cancer 6, 857–866 (November 2006)

miRNA-expression profiles classify human cancers.

Common miRNA genes differentially expressed in various cancers, suggesting common altered regulatory pathways ?

• miR-21 ; directly target the tumor suppressor PTEN in cholangiocarcinoma cells (Meng et al. 2006, Gastroenterology)

• mrR-21 ; knockdown in cultured glioblastoma cells triggers apoptosis by a caspase-dependent mechanism (Chan, J. A. et al. 2005, Cancer Res)

miR-21 as an anti-apoptotic and pro-survival factor

• Let-7 miRNA family ; Ras regulation(Voorhoeve, P. M. et al ,Cell, 2006)

• miR-372 and miR-373 ; facilitate the proliferation and transformation of cells

• miR-155 transgenic mouse in B-cells ; polyclonal pre-leukaemic pre-B-cell proliferation followed by B-cell malignancy(early event in oncogenesis that needs additional genetic alterations for the development of the fully malignant phenotype) (Costinean, S. et al. PNAS, 2006)

A new form of cancer predisposition?

• Abnormalities of miRNAs in the germline, an inherited predisposing event(blue person)• The overexpression of target oncogenes (red protein) in the case of miRNA deletion• The downregulation of target tumour-suppressor genes in the case of miRNA amplification (purple protein)

Short Summary• miRNAs located in genomic regions amplified in cancers function as

oncogenes, whereas miRNAs located in portions of chromosomes deleted in cancers function as tumour suppressors.

• Abnormal expression of miRNAs has been found in both solid and

haematopoietic tumours by various genome-wide techniques.

• The abnormally expressed miRNAs targeting essential protein-coding genes involved in tumorigenesis, such as the Ras by let-7 family members, the BCL2 anti-apoptotic gene by the miR-15a–miR-16-1 cluster,

• miRNA expression fingerprints correlate with clinical and biological

characteristics of tumors, including tissue type, differentiation, aggression and response to therapy.

• Germline sequence abnormalities in miRNA genes and targeted sequences in mRNAs .

miRNA & Breast Cancer

Numbers of publications on the relationship of Breast Cancer & miRNAs since 2005

87

9

0

20

40

60

80

100

Orginal Ariticale

Review

Numbers of publications according to research objective

16

5

45

37

0

10

20

30

40

50 Human BC Expression Profile

miRNA/target polymorphism

Functional Analysis

Genomic Localization

Method

miRNA expression profiles in Breast Cancer Pts. Tissue

• 76 breast cancers and 34 normal samples

• Deregulated expression of 29 miRNAs by microarray analysis

• A minimum predictive gene set of 15 candidates able to separate normal from cancer tissue

• The correlations bet. miRNA expression and standard pathologic features of breast cancers including lymph node status, vascular invasion, metastasis, ER, PR, and p53 status with the exception of ERBB2 expression

• Only a small set of 15 miRNA correctly predict the nature of the sample analyzed (i.e., tumor or normal breast tissue) with 100% accuracy.

Iorio, M. V. et al. Cancer Res 2005;65:7065-7070

Figure 1. Cluster analysis and PAM prediction in breast cancer and normal breast tissues

Deregulated expression of 29 miRNAs by microarray analysis

Deregulated expression of 29 miRNAs in tumor breast tissue

miRNA name Median expression ANOVA* P SVM prediction strength

PAM score Chromosome map

Cancer Normal Cancer Normal

mir-009-1 1.36 1.01 0.0091 8.05 0.011 –0.102 1q22

mir-010b 1.11 1.70 0.0449 8.70 –0.032 0.299 2q31

mir-021 1.67 1.08 0.0047 10.20 0.025 –0.235 17q23.2

mir-034 1.67 1.09 0.0106 8.05 0.011 –0.106 1p36.22

mir-102 (mir-29b) 1.36 1.14 >0.10 8.92 0.000 –0.004 1q32.2-32.3

mir-123 (mir-126) 0.92 1.13 0.0940 9.13 –0.015 0.138 9q34

mir-125a 1.20 1.73 0.0033 8.99 –0.040 0.381 19q13.4

mir-125b-1 1.30 2.87 0.0265 14.78 –0.096 0.915 11q24.1

mir-125b-2 1.26 2.63 0.0233 17.62 –0.106 1.006 21q11.2

mir-140-as 0.93 1.10 0.0695 11.01 –0.005 0.050 16q22.1

mir-145 1.52 3.61 0.0040 12.93 –0.158 1.502 5q32-33

mir-155 (BIC) 1.75 1.37 0.0012 10.92 0.003 –0.030 21q21

mir-194 0.96 1.09 >0.10 11.12 –0.025 0.234 1q41

mir-204 0.78 0.89 0.0022 8.10 –0.015 0.144 9q21.1

mir-213 3.72 2.47 0.0108 9.44 0.023 –0.220 1q31.3-q32.1

Differentially expressed miRNAs associated With biopathologic features of breast cancers

ER and PR

lymph node status, vascular invasion, metastasis, p53 status

What mRNA could be targeted by these targets?

• miR-10b targets ; FLT1(v-crk homologue), the growth factor BDNF, and the transducing factor SHC1

• miR-125b target ; YES, ETS1, TEL, and AKT3; the growth factor receptor FGFR2; mitogen-activated signal transduction pathway VTS58635, MAP3K10, MAP3K11, and MAPK14.

• miR-145 target ; cell cycle promoters such as cyclinsD2 and L1; and MAPK transduction proteins such as MAP3K3 and MAP4K4

• miR-21 target ; the TGF-beta gene

• miR-155 ; tumor suppressor genes SOCS1 and APC,

and the kinase WEE1, which blocks the activity of Cdc2

and prevents entry into mitosis. HIF1A.

Now, using PITA algorithms

Correlation analysis between DNA copy number alteration

and miRNA expression

miRNA & BC metastasis

miR-10b is highly expressed in metastatic breast cancer cells and positively regulates cell migration and invasion.

miR-10b induces tumour invasion.

Stromal Invasion

Muscular Invasion

Vascular Invasion

Ki-67 Ki-67MECA-32 MECA-32

miR-10b induces distant metastasis

Clusters of metastatic cells

miR-10b expression level is associated with the metastasis outcome in breast cancer patients

a. miR-10b expression level is associated with the metastasis outcome in breast cancer patients.

b. miR-10b suppresses HOXD10, leading to induction of RHOC.

HOXD10 : preveously reported to be progressively lost in BC showing increasing degree of malignancy(metastasis supression)

Clinical association of miR-335 and miR-126 with metastasis-free survival

20 primary breast tumor samplesmiR-335 and miR-126; metastasis suppressor miRNAs in human breast cancer

miRNA signatures classify breast cancer subtype

Expression profiling of 453 miRNAsperfomed in 29 early-stage breast cancer specimens

NA™ miChip array pl

atforms (Exiqon versi

on 7, containing 453

miRNA sequences)

• the genome-wide expression profiling of miRNAs in primary BC

• microarray containing 435 mature human miRNA oligonucleotide probes

• 9 miRNAs(miR-21, miR-365, miR-181b, let-7f, miR-155, miR-29b, miR-181d, miR-98, and miR-29c) up-regulated greater than 2 fold in BC compared with NAT

• 7 miRNAs (hsa-miR-497, hsa-miR-31, hsa-miR-355, hsa-miR-320, rno-mir-140, hsa-miR-127 and hsa-miR-30a-3p) down-regulated greater than 2 fold.

RNA. 2008 November; 14(11): 2348–2360.

miR-21, serve as a molecular prognostic marker for BC and disease progression

• TaqMan RT PCR in 113 BC tumors

• High level expression of miR-21

: with advanced clinical stage (P = 0.006), lymph node metastasis (P = 0.007), shortened survival of the patients (HR=5.476, P < 0.001)

Multivariate Cox regression analysis revealed this miR-21 (HR=4.133,P = 0.001) to be independent of disease stage (HR=2.226, P = 0.013) and histological grade (HR=3.681, P = 0.033)

Cancer Treatment Reviews 25 (2009) 328-334

MiRNA Polymorphisms & Breast Cancer

Prasun J Mishra‌et al Pharmacogenomics, Mar 2009

• 3 important aspects in analyzing miRNA target site mutation/polymorpism

• (i) functional: testing SNP-mediated differential miRNA targeting

• (ii) genetic: testing association of the SNP with a disease or quantitative trait and

• (iii) mechanistic: testing a mechanism by which the differential miRNA activity can lead to disease

• Recommend for the convincing evidence for all three components by

• (i) providing experimental (in vivo if possible) validation of SNP-mediated differential miRNA targeting

• (ii) minimizing or eliminating the confounding effects of population stratification

• (iii) explicitly testing an underlying mechanism by which the poly-miRTS could contribute to disease pathogenesis.

7

• Case-control study using a familial study population

• Study population : 1223 German breast cancer families(BRACA1/2 mutation negative) and 1495 unrelated German controls

• Sample : genomic DNA from peripheral blood lymphocytes collected during the years 1997–2007 by 7 centers of the German Consortium for Hereditary Breast and Ovarian Cancer

• SNP selection : 11 putative functional SNPs in miRNA target sites located in genes involved in cancer and breast cancer

• Genotyping : TaqMan allelic discrimination. Primers and TaqMan probes, 5 ng of genomic DNA per assay

• Conclusions : a variant in the ESR1 affecting a putative miRNA-binding site of miR-453 was significantly associated with familial breast cancer risk, especially in premenopausal and high-risk women

11 putative functional SNPs in miRNA target sites located in genes involved in cancer and breast cancer

Genotype frequencies polymorphisms in breast cancer-related genes in the German study population

Genotype frequencies of ESR1 polymorphism rs2747648 in the German study population

according age stratification and high-risk families

From the in silico analysis, C variant allele of miR-453 is stronger than T allele T allele the binding of miR-453 miRNA-mediated ESR1-repression ESR1 an increased breast cancer risk

: protective effect observed for the C allele is biologically reasonable.

• 42 pts. w/ familial breast cancer

• 23 of pts were BRCA1 mutation, 8 had BRCA2 mutation, 7 were non carriers of BRCA1/2

• 17 selected miRNA gene predicted to regulate key BC genes 7 novel genetic variants

• miR-30c-1 and miR-17 vatiantsin noncarriers of BRCA1/2

7 Novel genetic variants in selected miRNA genes in BC

17 miRNA genesPrecursur seq.

Genomic seq. PCR/ Sequencing

Sequence variations in the pri-miRNAcan translate into structural alterations

C(-48.1kcal/mol, more stable)T(-43.0kcal/mol, less stable)

conformational change in the predicted secondary structure(RNAHYbrid) altered exp.of mature miRNA

Effects of miR-17 on the luciferase reporter gene bearing 3’UTR segment from BRCA1

miRNA, as a new, novel, CANCER BIOMARKER ?

http://www.hammerstockblog.com/

miRNA being evaluated as therapeutic targets as well as diagnostic targets

Designed to Differentiate

bet. pancreatic cancer and

pancreatitis

Why miRNA better than mRNA as a diagnostic tool?

• Remarkably, the number of miRNAs analyzed was far smaller than the number of mRNA transcripts (≈200 miRNAs vs. ≈15000 mRNAs).

• More useful because of their regulatory role and their functions in modulating cellular differentiation.

Research Fields on miRNA,as a Breast Cancer Biomarker

• SUSCEPTIBILITY MARKER for BC miRNA gene variation/genomic alteration of miRNA genes – polymorphism in miRNA target gene/miRNA related gene– copy number variation in miRNA gene in breast cancer

• DIAGNOSTIC MARKER to BC- Deregulation of miRNA biogenesis related genes in breast cancer- Differential miRNA expression associated with biopathologic features of breast cancer[receptor status, TNM stages, vascular invasion status… etc.]

• PROGNOSTIC MARKER to BC– miRNA profiling / response to therapeutics / disease outcome

Small RNAs Are Raising Big Expectations

Karyn Hede Vol.101 Issue 12 June 16. 2009