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Chia-Cheng Chang, Ph.D.

Department of Pediatrics

and Human Development

Michigan State University

Epigenetic Mechanisms of Carcinogenesis

非基因突變的致癌機制

Epigenetic Mechanisms of Carcinogenesis

非基因突變的致癌機制

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Epigenetic Change

Alteration of gene expression at the transcriptional, translational or posttranslational levels.

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Some Common Causes of Altered Gene Expression

Methylation Methylation of DNAof DNA

Histone deacetylationHistone deacetylation

Altered stability of mRNAAltered stability of mRNA

PhosphorylationPhosphorylation of proteinsof proteins

TraffickingTrafficking

Protein binding/Protein binding/complexingcomplexing

CellCell--cell communicationcell communication

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Causes of Cancer

1. Endogenous mutagenesis: Chemical instability of DNA (e.g., depurination), oxygen free radicals produced in cellular metabolism, errors in DNA replication, developmental factors (e.g., lifetime exposure to estrogen).

2. Physical agents: UV, x-rays, alpha-particle (radon).3. Chemical carcinogens: Tumor initiators (mutagens)

and promoters (mitogens); tobacco and tobacco smoke, environmental pollutants, workplace exposures, diet, alcoholic beverages, etc.

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Causes of Cancer4. Microbes: viruses and bacteria (e.g., Helicobacter Pylori

in stomach cancer).Virus groups associated with human cancers:DNA viruses– Hepatitis B virus – Hepatocellular carcinoma– Human papilloma virus – Cancer of the cervix– Epstein-Barr virus – Lymphoma and

nasopharyngeal carcinoma

RNA viruses– Hepatitis C virus – Hepatocellular carcinoma– HIV 1 and 2 – Kaposi’s sarcoma

Non-Hodgkin’s lymphoma– HTLV 1 and 2 – Adult T-cell leukemia

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Viral Oncogene products Inactivate Tumor Suppressor Genes

SV40 large T-antigen - Rb and p53Adenoma virus Ela - Rb

Elb - p53Human papilloma virus 16

E7 - Rb; Inactivation of p16/RbE6 - p53; Activation of telomerase

Hepatitis B virus x protein inactivates p53

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Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transriptional activity, and association with transcription factor ERCC3Wang X. et al. Proc. Natl. Acad. Sci. 91:2230-2234, 1994.

Functional inactivation but not structural mutation of p53 causes liver cancerUeda H. et al. Nature Gent. 9:41-47, 1995

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Direct evidence for the participation of gap junction-mediated intercellular communication in the transmission of damage signals from alpha -particle irradiated to nonirradiated cells

Azzam EI. et al. PNAS. 98:473-8, 2001

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Membrane-derived Second Messenger Regulates X-ray-mediated Tumor Necrosis Factor α Gene InductionHallahan D.E. et alProc. Natl. Acad. Sci. USA 91:4897-4901,1994.

Treatment of human cells with ionizing radiation(or H2O2) was associated with the production of arachidonic acid, activation of PKC and induction of TNF-α. These effects can be abolished by phospholipase A2 inhibitors (dexamethasone, pentoxifilline)

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Epigenetic Mechanisms of Carcinogenesis

Production of genetically normal mosaic mice from malignant teratocarcinoma cells (Mintz and Illmense 1975).Some carcinogens (tumor promoters) are notgenotoxic.

Modification of gene expression, function due to developmental changes or exogenous factors could lead to cancer.

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Epigenetic Mechanisms of Carcinogenesis

DNA hypermethylation down-regulates tumor suppressor genes; DNA hypomethylation up-regulates oncogenes.Choline and methionine deficient diet cause hypomethylation of oncogene and liver tumor in rodents.Viral oncogene products inactivate tumor suppressor genes.

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Maintenance of DNA Methylation

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DNA Methylation and Gene Expression

Cytosine residues in 5’CpG are often postsynthetically

methylated

CpG methylation is involved in long-term silencing of

certain gene during development

The methyl-CpG-binding proteins MeCP1 and MeCP2

interact specifically with methylated DNA and mediate

transcriptional repression

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Goodman J.I. Watson R.E. Ann. Rev. Pharmacol. Toxicol.42:501-25,2002

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Multiple Factor Controlling DNA Methylation

Jay Goodman and Rebecca WatsonJay Goodman and Rebecca Watson

Ann. Rev. Ann. Rev. PharmacolPharmacol. . ToxicolToxicol. 42:501. 42:501--25,200225,2002

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Multiple Roles of DNA Methylation in Carcinogenesis

I. Epigenetic Change1. Hypermethylation-represses tumor

suppressor genes.2. Hypomethylation-activates oncogenes.

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Multiple Roles of DNA Methylationin Carcinogenesis

II. Mutation

1. Hypermethylation of CmC --> T hydrolytic deamination

2. C --> U Hpa II DNA (cytosine-S) methyltransferase

U --> T

3. Mark chromosome regions for deletion

4. Binding of methyltransferase decreases repair

For review see: Ann. Rev. Pharmacol. Toxicol. 42:501-25,2002

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Hhal and Hpall DNA methyltransferase bind DNA mismatches, methylate uracil and block DNA repairYang A.S. et al. Nuclic acid Res. 23:1380-1387,1995.

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Initation and Cell Proliferation in Multistage Carcinogenesis

Swenberg et al. Environ. Health Perspect 76:57-64,1987.

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A Genetic Model for Colorectal Tumorigenesis

CHROMOSOME ALTERATION

GENE

5q mutation or loss FAP

DNA Hypomethylation

12p Mutation

K-Ras

NORMAL EPITHELIUM

HYPERPROLIF EPITHELIUM

EARLY ADENOMA

INTERMEDIATE ADENOMA

LATE ADENOMA

CARCINOMA METASTASIS

18q Loss

DCC?

Other Alterations

17p Loss p53

Cell 61: 759-767, 1990

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DNA Methylation and CarcinogenesisHypomethylation

Human tumors normally exhibit decreased overall genome methylation

Cancer Res. 48:1159-1161, 1988; 64:4238-4243,2001Adv. Cancer Res. 54:1023,1990Gene. Cytogenet. 97:83-89,1987

Hypomethylation occurs relatively early in carcinogenesis

New Engl J./med. 319:525-532,1988.Cancer Res. 48:1159-1161, 1988Gut 39:434-438, 1996.

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[CANCER RESEARCH 61, 4238–4243, May 15, 2001]Genome-wide Hypomethylation in HepatocellularCarcinogenesis1

Chiu-Hung Lin, Sen-Yung Hsieh,2 I-Shyan Sheen, Wei-Chen Lee, Tse-Ching Chen, Wei-Chue Shyu, and Yun-Fan LiawLiver Research Unit [S-Y. H., I-S. S., W-C. S., Y-F. L.], Departments of General Surgery [W-C. L.], and Pathology [T-C. C.], Chang Gung Memorial Hospital, Taipei, Taiwan155, and Department of Microbiology and Immunology, Chang Gung University School of Medicine, Taoyuan 333 [C-H. L., S-Y. H.], Republic of Taiwan

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Genome-wide Hypomethylation in Hepatocellular Carcinogenesis

Chiu-Hung Lin et al., Cancer Res 64:4238-4243, 2001

Compared with the paired non-HCC liver tissues, genome-wide 5-methylcytosinecontent in HCC was reduced in all of the tested HCC samples (17). Genome-wide 5-methylcytosine content did not significantly differ among normal, non-cirrhotic and cirrhotic liver tissues.

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In the Colon

A significant reduction in 5- methylcytosinecontent in adenoma and adenocarcinomacompared with normal, paired colonic mucosa, whereas no difference existed between benign adenoma and malignantadenocarcinoma.

Feinberg A.P. et al. Cancer Res.48:1159-1161, 1988.

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In the Stomach

Global DNA hypomethylation occurs inthe early stages of neoplastic transformationwith no further change of DNAhypomethylation in gastric carcinoma.Cravo, M. et al. Gut 39:434-438, 1996.

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A SAGE(Serial Analysis of Gene Expression) View of Breast Tumor ProgressionD. A. Porter et al. Cancer Res. 61:5697-5702,2001.

The most dramatic and consistent The most dramatic and consistent phenotypic change occurred at the phenotypic change occurred at the normalnormal--toto--in situ carcinoma transitionin situ carcinoma transition

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DNA Methylation and Carcinogenesis

Hypermethylation Tumor Suppressor genes hypermethylated in human cancer

Cancer Res. 61:3225-3229,2001.

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A Gene Hypermethylation Profile of Human Cancer M. Esteller et al. Cancer Res.61:3225-3229, 2001.

Analyzed promoter hypermethylation changes in 12 genes from 600 primary tumor samples representing 15 major tumor.P16INK4a, P15INK4b － Cell cycle regulation BRCA1, hMLH1, MGMT － DNA repairTIMP3, CDH1, DAPK － Cell adherence, metastasisGSTP1 － Metabolic enzymeP14ARF, P73 － P53 networkAPC － β - catenin function

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A unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and others are cancer-type specific

P16 INK4a － Many tumor typesAPC － Liver, pancreas, stomachP14 ARF － Colon, stomachGSTP － Liver, breast, prostateMGMT － Colon, brainP15 INK4b － Leukemia

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Simultaneous inactivation of multiple pathway

Colon : P16 INK4a, hMLH1, TIMP3Breast : P16 INK4a, BRCA1, CDH1Lung : P16 INK4a, MGMT, DAPK

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DNA Methylation and Carcinogenesis

Decreased availability of methyl group caused liver Decreased availability of methyl group caused liver tumors in rodents. Diet devoid of tumors in rodents. Diet devoid of cholinecholine and and deficient in deficient in methionine methionine leads to :leads to :

1.1. Increased Increased hepatocyte hepatocyte proliferationproliferation2.2. HypomethylationHypomethylation and increased expression of and increased expression of

oncogenes oncogenes (reversible)(reversible)3.3. Liver tumor formationLiver tumor formation

See review by Jay Goodman and Rebecca WatsonSee review by Jay Goodman and Rebecca WatsonAnn. Rev. Ann. Rev. PharmacolPharmacol. . ToxicolToxicol. 42:501. 42:501--25,200225,2002

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S-Adenosyl methionine (SAM) MetabolismAm. J. Nutrition 55:131-138,1992

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Arsenic is Classified as a Human Carcinogen

Int. Agency for Cancer Res. (IACR)Int. Agency for Cancer Res. (IACR)United StatesUnited States

Environmental Protection Agency(EPA)Environmental Protection Agency(EPA)Occupational Safety and Health Occupational Safety and Health Administration(OSHA)Administration(OSHA)National Toxicology Program(NTP)National Toxicology Program(NTP)

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Arsenic is a Human Carcinogen

Primary TargetPrimary Target------ SkinSkinHyperkeratosis of soles and palmsHyperkeratosis of soles and palmsBasal and Basal and squamous squamous cell carcinomacell carcinomaNonNon--sun exposed areasun exposed area

Secondary TargetSecondary Target------ liver, bladder, kidneyliver, bladder, kidneyLung cancer from inhalationLung cancer from inhalationNo animal modelNo animal model

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Source of Exposure

Drinking waterDrinking waterNaturally occurringNaturally occurringIndustrial contaminationIndustrial contamination

OccupationalOccupationalMining and weldingMining and weldingSemiconductor, glass and paper industrySemiconductor, glass and paper industry

MedicalMedicalpsorisispsorisis, , promyecyticpromyecytic leukemia(arsenic trioxide)leukemia(arsenic trioxide)

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Areas with High Arsenic Exposure

IndiaIndiaBangladeshBangladeshTaiwanTaiwanMexicoMexicoPart of S. America(Chile, Argentina)Part of S. America(Chile, Argentina)Part of U.S.(Western States. Michigan)Part of U.S.(Western States. Michigan)

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Carcinogenicity of Arsenic

NonNon--mutagenicmutagenicPotential DNA damaging agentPotential DNA damaging agentClastogenic Clastogenic actionaction

Effect of exposure are irreversibleEffect of exposure are irreversibleLong latent periodLong latent periodIncreases DNA Increases DNA hypomethylation hypomethylation in micein mice

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Sodium arsenite adminstration via drinking water increases genome-wide and Ha-ras DNA hypomethylation in methyl-deficient c57BL/6J miceR.S. Okoji et al. Carcinogenesis 23:777-785,2002

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Effect of Sodium Arsenite and Methyl-Deficiency on Methylation of Ha-ras

At 474/976 segmentAt 474/976 segment5/11 restriction sites 5/11 restriction sites hypomethylatedhypomethylated

At 962/1487 segmentAt 962/1487 segment

¼ restriction sites restriction sites hypomethylatedhypomethylated

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Mechanisms by which Arsenic Affects DNA Methylation

1.1. Depletion of methyl donor poolDepletion of methyl donor pool2.2. Inhibiting DNA Inhibiting DNA methyltransferasemethyltransferase3.3. Divert intracellular methyl donor to the Divert intracellular methyl donor to the

methylation methylation of arsenicof arsenic

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Smoking

Nicotineaddiction

Carcinogens(PAH, NNK etc)

Metabolicactivation

DNAadducts

Mutations(p53, K-ras etc)

Metabolic detoxification(GSTM1 etc)

Excretion NormalDNA

LungCancer

Repair

Cell death

Apoptosis

From Dr. Wen-Chun Hung lecture slide

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Pattern of p53 G T transversionsin lung cancers reflect the primary mutagenic of DNA –damage by tobaco smokeHainaut et al. Carcinogenesis 22:366-

374, 2001

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Regulation of Human Pancreatic Cancer Cell Growth by Tobacco-specific nitrosamine NNK

ß-blockerICI 118, 551

Inactivelipase

Activelipase

Phospholipids

Arachidonic acid5-lipox mk886

ThromboxanesProstaglandins

Leukotrienes

Cell growth

CoxInhibiter Cox

Weddle, D.L. et al., Carcinogenesis 22: 473-479, 2001

Stimulus:β-adrenergic agonist

NNK

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Polycyclic aromatic hydrocarbon with bay-like regions inhibited gap-junctional intercellular communication and stimulated MAPK activityRummel A.M. et al.

Toxicol Sci. 49:232-240,1999.

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Global View of Taiwan Value謝長廷：我覺得這件事(宋七力事件)對我最大

的傷害是社會突然用一種唯物論，實證主義去打擊所有相信宇宙神秘力量的人。

Hippocrates ( Father of Medicine)：Men think epilepsy divine merely because they donot understand it. But if they called everythingdivine which they do not understand, there wouldbe no end of divine things.

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謝長廷：我不是去賭博或墮落(指崇拜宋七力)，我是追求生命的價值。

Where ignorance is bliss, tis folly to be wise.(當無知會帶來喜悅，愚蠢人也變聰明)－ poet Thomas Gray

真的如此嗎？

It is morally as bad not to care whether a thing is true or not, so long as it makes you feel good as it is not to carehow you get your money as long as you have got it.－Edmond Way Teale

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Instead of acknowledging that in manyareas we are ignorant, we have intendedto say things like the universe is permeatedwith the ineffable.

在很多地方，我們寧願相信宇宙充滿不可思議的事情而不承認自己的無知。