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EPIGENETIC AND MICRORNA CIRCUITS IN CANCER

Dimitrios Iliopoulos, Ph.D

COURSE: BIOLOGY OF CANCER

EPIGENETICS

Definition: heritable changes in gene regulation that occur without a change in DNA sequence

Orchestration of key biological processes

•Tissue differentiation

•Developmental stages

•Aging

•Diseases (cancer)

Tumor suppressor genes

DNA methylation

Discuss…

•How could we detect a CpG island in a gene?

•Compare genetic and epigenetic dynamics

•Detection methods of DNA methylation

•Genes frequently methylated in cancers

•DNA methylation is applicable to medicine?

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DNA methylation

Discuss…

•How could we detect a CpG island in a gene?

•Compare genetic and epigenetic dynamics

•Detection methods of DNA methylation

•Genes frequently methylated in cancers

•DNA methylation is applicable to medicine?

CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

CpG island: An area with high frequency of CpG sites

CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

?

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CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

?

Regulatory area

100%

50%

% C

pGsite

s

CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

CpG island

> 60% CpG sites

50%

100%

Regulatory area

% C

pGsite

s

Question 1

2 genes in their regulatory area they have 11 CpG sites

The first one has a CpG island, what about the second one?

Keyword: Density of CpG sites

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CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

CpG island11 CpG sites

Exon 1 Exon 2 Exon 3

11 CpG sites No CpG island

CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

CpG island 1 CpG island 2

Exon 1 Exon 2 Exon 3

CpG island 1

CpG Island Searcher

Select the lower limit values

Gap between adjacent islands

Length

Obs/ExpCpG

%GC

Submit Reset

60%59%58%57%56%55%54%53%52%51%50%

0.700.690.680.670.660.650.640.630.620.610.60

900bp800bp700bp600bp500bp400bp300bp200bp

300bp250bp200bp150bp100bp

Sequence

http://www.uscnorris.com/cpgislands2/cpg.aspx

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CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

ACTIVE STATE

CpG AREA IN REGULATORY GENE REGIONS

Exon 1 Exon 2 Exon 3

REPRESSED STATEEnzymes

•DNMT 1 (maintenance of DNA methylation)

•DNMT 3a, 3b (de novo DNA methylation)•DNMT 3L (establishment of maternal imprints)

Question 2

Exon 1 Exon 2 Exon 3

Exon 1 Exon 2 Exon 3

Methylation in 1 CpG site could affect gene expression?

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DNA methylation

Discuss…

•How could we detect a CpG island in a gene?

•Compare genetic and epigenetic dynamics

•Detection methods of DNA methylation

•Genes frequently methylated in cancers

•DNA methylation is applicable to medicine?

DYNAMICS OF EPIGENETIC SILENCING

A somatic genetic mutation is associated with an immediate block in the production of a

functional protein

Epigenetically mediated silencing might begin gradually, starting in the earliest phase

of tumor progression

DNA methylation

Discuss…

•How could we detect a CpG island in a gene?

•Compare genetic and epigenetic dynamics

•Detection methods of DNA methylation

•Genes frequently methylated in cancers

•DNA methylation is applicable to medicine?

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DNA methylation detection methods

DNA Bisulfite treatment MSP

C CH3

C

C

T

Sequencing

ATCGCGTATTACGTG

Example

3 CpG sites

DNA methylation detection methods

DNA Bisulfite treatment MSP

C CH3

C

C

T

ATCGCGTATTACGTG

Sequencing

ATCGCGTATTACGTG

CH3 CH3 CH3Example

If methylated…

DNA methylation detection methods

DNA Bisulfite treatment MSP

C CH3

C

C

T

ATCGCGTATTACGTG

Sequencing

ATTGTGTATTATGTG

Example

If unmethylated…

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Question 3

Tissue DNA extraction Bisulfite treatment MSP

40 breast cancer tissues

12 normal mammary tissues

Question: Detect methylation status of “favorite” gene?

Methodology?

• 38 / 40 methylated breast cancer tissues

• 11 / 12 unmethylated normal mammary tissues

Question 4

• 38 / 40 methylated breast cancer tissues

• 11 / 12 unmethylated normal mammary tissues

“Favorite” gene is regulated by DNA methylation in cancer.

What about the expression of “Favorite” gene?

Method of detection?

Correlation with DNA methylation?

DNA methylation

Discuss…

•How could we detect a CpG island in a gene?

•Compare genetic and epigenetic dynamics

•Detection methods of DNA methylation

•Genes frequently methylated in cancers

•DNA methylation is applicable to medicine?

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CHROMOSOMAL EPIGENETIC CANCER MAP

11 genes (2001)

DNA Methylation Society

CHROMOSOMAL EPIGENETIC CANCER MAP

28 genes (2005)

DNA Methylation Society

CHROMOSOMAL EPIGENETIC CANCER MAP

Estimation: 51 genes (2007)

DNA Methylation Society

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IMPORTANT EPIGENETICALLY REGULATED CANCER GENES

Gene name Map Cancer Types

APC 5q21 Colon, gastric, esophagealAR Xq11 ProstateBRCA1 17q21 Breast, ovarianER 6q25 Breast, ovarian, liver, lungMLH1 2p22 Colon, endometrial, gastric

MGMT 17q21 Brain, colon, lung, breastp14/ARF 9p21 Colon

p16 (CDKN2A) 17q21 Lung, colon, prostate, lymphomasRASSF1A 3p21 LungRb 13q14 Retinoblastomas, pituitary adenomas

DNA methylation

Discuss…

•How could we detect a CpG island in a gene?

•Compare genetic and epigenetic dynamics

•Detection methods of DNA methylation

•Genes frequently methylated in cancers

•DNA methylation is applicable to medicine?

DNA Methylation Cancer Markers

Why there is a need for prognostic, diagnostic markers?

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Question 5

Why there is a need for prognostic, diagnostic markers?

DNA Methylation Cancer Markers

DNA methylation would be a better marker than the markers available today in the clinic?

•2006 AACR meeting in Washington, DC several markers used today in the clinic are not so reliable as we believed (example PSA)

•DNA methylation is detected in early stages of carcinogenesis and usually in precancerous or normal tissues.

•Easily detected, cheap and accurate methods.

Why there is a need for prognostic, diagnostic markers?

Early detection of cancer More effective therapies

MODEL: DNA Methylation in Lung Cancer

•More than 1.5 million people will die from lung cancer in the next 5 years

• Most of the lung cancer cases are detected in the late stages of the disease.

•Cancers that diagnostic tests have been developed reveal better survival rates.

•It will be very important to detect lung cancer in early stages (I,II).

•Difficulty for biopsy but easily accessible material (sputum).

•Detection of DNA methylation markers in sputum

•Candidates for this test: smokers for >20 yo – 2 packs daily.

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DNA Methylation Affects Cancer Therapies

•Case 1: 37 yo female with breast cancer (stage II) and ERa+, HER3+.

Treatment

Surgery

Chemotherapy

Adjuvant therapy

2007: Tamoxifen for 5 years and Herceptin for 1 year

Risk of this therapy: tamoxifen resistance

positive: tamoxifennegative: aromatase inhibitor

2010: DNA methylation marker predict tamoxifen response

Question 6

•Case 2: 46 yo male with glioblastoma (Stage II).

Are you going to treat this patient with Temodal?

TreatmentSurgery

Radiation therapy

Chemotherapy (Temodal)

MGMT methylation status predicts temodal response

DNA Methylation Affects Cancer Therapies

•Case 2: 46 yo male with glioblastoma (Stage II).

Treatment

Surgery

Radiation therapy

Chemotherapy (Temodal)

2005: Temodal (100 mg/m2) for 8 cycles (5 days – 28 days)

2007: MGMT methylation status predicts temodal responsepositive: Temodalnegative: No chemotherapy

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HISTONE MODIFICATIONS

HISTONE MODIFICATIONS

HISTONE MODIFICATIONS

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EPIGENETIC CHANGES IN CANCER

Question 7

Why DNA methylation inhibits gene expression?

How histone modifications affect gene expression?

EPIGENETIC CHANGES IN CANCER

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STRATEGIES FOR EPIGENETIC THERAPY

Epigenetic therapy with DNA methylation and HDAC inhibitors is now a reality. While these agents are approved as single agents,combination therapies are likely to gain traction in the future.

Clinical trials with DNMT and HDAC inhibitors

Target Drug Clinical trials

DNA methylation Decitabine Phase III

Histone deacetylation Phenylbutyric acidSAHA

Valproic acid

Phase I/IIPhase II/IIIPhase I/II

Clinical trial in Harvard University

Decitabine and Valproic acid in treating patients with non small cell lung cancer

Protocol: Patients will receive decitabine over 1 hour on days 1- 10 and oral valproic acid three times daily on days 5-21. Treatment will be

repeated every 28 days for 6 courses.

NEW PLAYERS IN EPIGENETIC REGULATION

DNA METHYLATION

HISTONE MODIFICATIONS

RNA

INTERFERENCE

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NEW PLAYERS IN EPIGENETIC REGULATION

DNA METHYLATION

HISTONE MODIFICATIONS siRNA

NEW PLAYERS IN EPIGENETIC REGULATION

DNA METHYLATION

HISTONE MODIFICATIONS shRNA

NEW PLAYERS IN EPIGENETIC REGULATION

DNA METHYLATION

HISTONE MODIFICATIONS microRNA

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Biogenesis of microRNAs

High mRNA levels but low protein levels (post-transcriptional regulation)Low mRNA levels and low protein levels

Slack et al. Nat Rev Cancer, 2006

microRNAs as tumor suppressors/oncogenes

Tumorsuppressor Oncogene

The challenge of identifying microRNA targets

target 5' C GGUG UA A 3'CUGCUU UG GGUUCCGUGACGAA AC CUAAGGCA

microRNA 3’ UC A C AC 5'

A microRNA binds in the 3’UTR of the target gene

Incomplete complementarity allows short stretches of mismatched base-pairs and G-U pairing, the identification of gene targets with a simple BLAST is impossible.

miRanda is an algorithm for finding genomic targets for microRNAs. This algorithm has been written in C and is available as an open-source method under the GPL. MiRanda was developed at the Computational Biology Center of Memorial Sloan-Kettering Cancer Center.

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The challenge of identifying microRNA targets

target 5' C GGUG UA A 3'CUGCUU UG GGUUCCGUGACGAA AC CUAAGGCA

microRNA 3’ UC A C AC 5'

A microRNA binds in the 3’UTR of the target gene

“miRNA seed”

•Several studies have shown that the 5’end of the microRNA is crucial for the stability and proper loading of the microRNA in the miRISC complex and biological function.

• A single microRNA might bind as many as 200 targets and these targets can be diverse in their function

MicroRNA targets

Human miRNA targets

Search for your favorite gene/miRNA and view their targets

Drosophila microRNA targets

Search for your favorite gene/miRNA and view their targets

Zebrafish microRNA targets

Search for your favorite gene/miRNA and view their targets

http://www.microrna.org

MicroRNA targets

Human miRNA targets

Search for your favorite gene/miRNA and view their targets

Drosophila microRNA targets

Search for your favorite gene/miRNA and view their targets

Zebrafish microRNA targets

Search for your favorite gene/miRNA and view their targets

http://www.microrna.org

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MicroRNA targets

Query by microRNA(s):

Enter list of miRNAs

Display:All targets

Common targets

Query by gene(s):

Enter list of genes

Display:microRNA regulators of any of these genesmicroRNA regulators common to all of these genes

GO

MicroRNA targets

Query by microRNA(s):

hsa-miR-373

Display:All targets

Common targets

Query by gene(s):

Enter list of genes

Display:microRNA regulators of any of these genesmicroRNA regulators common to all of these genes

GO

MicroRNA targetsGenes targeted by: hsa-mir-373

[Download Results (Excel Format)]

Found 45 genes.Sorted from highest to lowest scoring Prepend "ENSG00000" to gene in summary table to get ENSEMBL gene id. 0

1181Histone acetyltransferase type B

subunit 2 (RBBP-7). ENST00000330735RBBP7ENSG00000102054hsa-miR-373

8291

RING finger protein 27 (Glioblastoma-expressed ring finger protein) (Tripartite motif-containing protein 8).

ENST00000302424TRIM8ENSG00000171206hsa-miR-373

10451

Orphan nuclear receptor NURR1 (Transcriptionally inducible nuclear receptor).

ENST00000339562NR4A2ENSG00000153234hsa-miR-373

19471ENSEMBL: similar to F10G7.10.p.

[RefSeq:NM_172070]ENST00000272793ENSG00000144357hsa-miR-373

Locations (numbering is relative to the start of the 3' UTR)

Number of HitsGene Description

EnsemblTranscript Id

Hugo Gene IdEnsembl Gene IdmiRNA

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MicroRNA targets

Query by microRNA(s):

Enter list of miRNAs

Display:All targets

Common targets

Query by gene(s):

Enter list of genes

Display:microRNA regulators of any of these genesmicroRNA regulators common to all of these genes

GO

MicroRNA targets

Query by microRNA(s):

Enter list of miRNAs

Display:All targets

Common targets

Query by gene(s):

CDKN2A

Display:microRNA regulators of any of these genesmicroRNA regulators common to all of these genes

GO

MicroRNA targets

Human miRNA target sites for ENSG00000147889 (CDKN2A)

ENSG00000147889 (CDKN2A): ENSEMBL: Cyclin-dependent kinase 4 inhibitor A (CDK4I) (p16-INK4) (p16-INK4a) (Multiple tumor suppressor 1) (MTS1). [SWISSPROT:P42771]

Scroll to the right to see details of miRNA target sites.Click on miRNA names to see details of miRNA/target sequence matches

1:hsa-miR-337

5' GGAGCUGGGCCAGCGCG

GGUUCAGGUCCAGCGCG 5’

Human miRNA: hsa-miR-337Score: 158, Energy: -26.7 kCal/mol

Conservation: 97.0%

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Microarray technology for microRNA profilingVolinia et al, PNAS 103(7):2257- 61, 2006.

540 samples 363 cancer samples

177 normal controls

Key issue: The level of differential expression that can be considered biologically significant.

36 over-expressed

21 down-regulated

Microarray technology for microRNA profilingVolinia et al, PNAS 103(7):2257- 61, 2006.

540 samples 363 cancer samples

177 normal controls

Key issue: The level of differential expression that can be considered biologically significant.

36 over-expressed

21 down-regulated

miR-21

PTEN

let-7

Ras

Microarray technology for microRNA profilingVolinia et al, PNAS 103(7):2257- 61, 2006.

540 samples 363 cancer samples

177 normal controls

Question 8: Potential therapy?

36 over-expressed

21 down-regulated

miR-21

PTEN

Therapy: Block miR-21 expression by antagomiRs

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Microarray technology for microRNA profilingVolinia et al, PNAS 103(7):2257- 61, 2006.

540 samples 363 cancer samples

177 normal controls

Question 9: Potential therapy?

36 over-expressed

21 down-regulated

let-7

Ras

Therapy: Overexpress let-7

MicroRNA expression in human cancers

Gene

Same type of cancer

DNA methylation Histone modifications microRNAs

MECHANISMS REGULATING GENE EXPRESSION

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Cancer Traditional Treatment

Chemotherapeutic agents

Cancercells

Noncancerous cells

Deadcancercells

Dead noncancerous cells

Cancer Nanotherapy

Nanodevices

Cancercells

Noncancerous cells

Deadcancercells

Intact noncancerous cells

Nanodevices of the future

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Biological Engineering Division of Massachusetts Institute of Technology

Dr Sasisekharan Lab

Nanocell is the first nanodevice capable of transferring two drugs

Nanoparticles

(e.g WWOX plasmid)

Phospholipid membrane coating

Drug encapsulated in lipid layer (e.g Decitabine)

Intra-tracheal Aerosol Delivery

Nanocell

Nanotechnology in patient care

Today 2020

Epigenetic mechanisms of gene regulation

Modulation of gene expression

BIOMEDICAL RESEARCH

DNA methylation, histone modifications

microRNA deregulation

Drug sensitivity

Animal studies

Clinical studies

IN V

ITRO

IN V

IVO

Cancer Markers

Animal studies

Clinical studies

THERAPEUTIC AGENTS DIAGNOSTIC MARKERS