epigenetic and microrna circuits in cancer dimitrios...

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1 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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  • 1

    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?

  • 2

    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

    ?

  • 3

    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

  • 4

    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

  • 5

    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?

  • 6

    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?

  • 7

    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…

  • 8

    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?

  • 9

    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

  • 10

    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?

  • 11

    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.

  • 12

    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

  • 13

    HISTONE MODIFICATIONS

    HISTONE MODIFICATIONS

    HISTONE MODIFICATIONS

  • 14

    EPIGENETIC CHANGES IN CANCER

    Question 7

    Why DNA methylation inhibits gene expression?

    How histone modifications affect gene expression?

    EPIGENETIC CHANGES IN CANCER

  • 15

    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

  • 16

    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

  • 17

    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.

  • 18

    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

  • 19

    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

  • 20

    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%

  • 21

    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

  • 22

    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

    ?

  • 23

    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

  • 24

    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