Notes on the epigenetic (transplacental and epigenetic (transplacental and transgenerational) origins transgenerational) origins of childhood cancerchildhood cancer

ERNESTO BURGIOECERI - European Cancer and Environment Research InstituteISDE Scientific Committee

(1) Cancer continuous increase(1) Cancer continuous increase

Cancer is generally associated with old age and its continuous increase, observed throughout the 20th century in all the industrialized countries, is often explained

through the theory of the progressive accumulation of oxidative and stochastic genetic damage (SMT)

and through the continuous improvement of our diagnostic capacities.

The fact that this increase, from the end of the 80s to 2000, has involved individuals of all ages, young people included, has been too often underestimated

FOREWORD 1 FOREWORD 1

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the significant increase significant increase in the in the Less Developed CountriesLess Developed Countries & in young people all overin young people all overthe worldthe world demonstrates the limits of the SMT (limits of the SMT (necessary link necessary link between between agingaging & &CA)CA)

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(2) Child cancer increase(2) Child cancer increase

Child cancers Child cancers are generally considered as a rare disease. ButBut it is worth rememberingworth remembering

• that, statistically, about 1 in 5-600 children about 1 in 5-600 children will develop cancer before the age of 15;

• that, in spite of the decisive improvement in diagnosis and therapy in the last decades, cancer is is the leading cause of death due to diseases the leading cause of death due to diseases among children over the first year of ageamong children over the first year of age;

• that, even at this age, a continuous and significant increase has been seen during the last decades.

Incidenza di tumori (anno/100.000)Incidenza di tumori (anno/100.000)

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Leucémie Leucémie lymphatilymphatiqueque

Leucémie lymphatiqueLeucémie lymphatiqueEncéphaleLymphômesNeuroblastoma-Retinoblastoma1

<1 Tessuti molli, rene (Wilms), gonadi

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It is generally argued that childhood cancers are a rare condition.

But it should be reminded

that CANCER is the main cause of death by disease in childhood

that there is a constant and significant increase of tumors in the world for this age group

that 1 : 5-600 children falls ill with cancer

That more than 13 000 children fall ill

with cancer each year in the U.S.

Bleyer A, O’Leary M, Barr R, Ries LA,editors. Cancer epidemiology in older adolescents and young adults 15-29 ears of age, including SEER incidence and survival: 1975-2000. NIH Pub. No.06-5767. Bethesda (MD): NationalCancer Institute; 2006. Jemal A, SiegelR, Ward E, et al. Cancer statistics,2008. CA Cancer J Clin 2008;58:71 – 6.

Alberto Tommasini, Laboratorio Immunologia Pediatrica, IRCCS Burlo Garofolo

is is the leading cause of death due to the leading cause of death due to diseases among children over the first year diseases among children over the first year of ageof age

(3) ACCIS(3) ACCIS

• The recent IARC reportsIARC reports of a significant increase in childhood cancer in Europe and especially in Italy has caused concern,

• forcing us to reconsider critically the forcing us to reconsider critically the dominant model of carcinogenesis. dominant model of carcinogenesis.

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Cancer incidence in childhood and adolescence - EUROPE( 1970-1999)( 1970-1999)

mother

latency

A first draft of the report, published on the Lancet in 2004, demonstrates an annual increase of 1-1,5% for all cancers (with more marked increases in lymphomas, soft tissue sarcomas, tumors of the nervous system…) .

As we may easily argue from the recent project ACCIS (Automated Childhood Cancer Information System) - a comprehensive monitoring conducted by a team of epidemiologists IARC on 63 cancer registries from 19 European countries, for a total of over 130 thousand tumors of all types (113 thousand children and 18 thousand teenagers)

Steliarova-Foucher E, Stiller C, Kaatsch P, Berrino F, Coebergh JW, Lacour B, Parkin M. Geographical patterns and time trends of cancer incidence and survival among children and adolescents in Europe since the 1970s (the ACCISproject): an epidemiological study. Lancet. 2004 Dec 11-17;364(9451):2097-105

http://www-dep.iarc.fr/accis.htm

These data should not be should not be underestimated underestimated for at least for at least 4 reasons:4 reasons:

These data should not be should not be underestimated underestimated for at least for at least 4 reasons:4 reasons:

..in the last 20 years (1978 e il 1997) the overall incidence rate has increased significantly with an average annual percentage change (AAPC) of 1,1% (> 2% in the first year; 1,3 % during adolescence).

• the large size of the study sample large size of the study sample (63 cancer registries from 19 European countries, for a total of more than 130000 cancers of all kinds: 113000 strictly paediatric and 18000 teenager cancers);

• a sufficiently prolonged period of observation (20 years); sufficiently prolonged period of observation (20 years); • the the maximum increase in the first year of agemaximum increase in the first year of age, which

suggests a transplacentaltransplacental (from maternal and fetal exposure to pro-carcinogenic agents) or even a or even a transgenerationaltransgenerational ((epigenetic/gameticepigenetic/gametic) origin; ) origin;

• the concomitant increaseconcomitant increase in the whole northern hemisphere of a variety of chronic degenerative diseases chronic degenerative diseases (endocrine-metabolic: obesity, type 2 diabetes; immune-mediated: allergies, autoimmune diseases, neuro-development and neuro-degenerative diseases: autism, ADHD, Alzheimer's disease.. ) for all of whichfor all of which a significant pathogenic role of the mechanisms mechanisms of early epigenetic dysregulation (of early epigenetic dysregulation (fetal programmingfetal programming) ) on various organs and tissues on various organs and tissues has recently been suggestedhas recently been suggested ((DOHaD-Developmental Origins of Health and DiseasesDOHaD-Developmental Origins of Health and Diseases).).

Gluckman PD, Hanson MA. Developmental origins of disease paradigm: a mechanistic and evolutionary perspective. Pediatr. Res. ( 2004); 56:311–17

Do the somatic cells of a single tissue undergo regression for intrinsic, accidental reasons (mutations or epi-mutations) and de-differentiate …

What is Cancer ?

Is cancer a Genetic Disease ?

Is cancer a Genetic Disease ?

Over your lifetime, random gene changes are passed along as your body cells grow and divide, so they accumulate

Nature

The Somatic Mutation Theory of Carcinogenesis

Chemicals and radiation act by damaging genes, viruses introduce their own genes into cells, and heredity passes on alterations in genes that make a person more susceptible to cancer

Genes are altered, or "mutated," in various ways as part of the mechanism by which cancer arises.

The Somatic Mutation Theory of Carcinogenesis

Tumor development proceeds via a process formally analogous to Darwinian evolution, Darwinian evolution, in which a succession of stochastic mutations succession of stochastic mutations, each conferring one or another type of growth advantage, leads to the progressive conversion of normal human cells into CA-cells…

CA-cells have CA-cells have defects in regulatory circuits that defects in regulatory circuits that govern normal cell proliferation and homeostasisgovern normal cell proliferation and homeostasis… the vast catalog of CA-cell genotypes is a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth:

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We suggest that the vast catalog of cancer cell genotypes is a manifestation of six essential alterations in cell physiology that collectively dictate malignant growth

What’s Cancer ? 1

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RTK Receptor

Tyrosine Kinases

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Alterations in three types of genes are responsible for tumorigenesis: oncogenes, tumor-suppressor genes and stability genes Unlike diseases such as cystic fibrosis or muscular dystrophy, wherein mutations in one gene can cause disease, no single gene defect 'causes' cancer. Mammalian cells have multiple safeguards to protect them against the potentially lethal effects of cancer gene mutations, and only when several genes are defective does an invasive cancer develop

The revolution in cancer research can be summed up in a single sentence: cancer is, in essence, a genetic disease

What’s Cancer ? 2

Vogelstein B, Kinzler KW. Cancer genes and the pathways they control Nat Med. 2004 Aug;10(8):789-99.Vogelstein B, Kinzler KW. Cancer genes and the pathways they control Nat Med. 2004 Aug;10(8):789-99.

The GWAS efforts are certainly creating bigger haystacks …

In a recent editorial on NatureHeidi Ledford stated that the millions of genetic sequences and SNPs accumulated in an attempt to decipher the genetics of cancer have built giant haystacksin which researchers have gone lost …

The full genome sequence of a lung cancer cell line, for example, yielded 22,910 point mutations, only 134 of which were in protein-coding regions

The full genome sequence of a lung cancer cell line, for example, yielded 22,910 point mutations, only 134 of which were in protein-coding regions

• .. there is now ample evidence that some specific epigenetic alterations, (primarily the hypomethylation of DNA, with activation of oncogenes and increased mobility of mobile sequences) ** are the result of protracted genomic are the result of protracted genomic stressstress (eg (eg chronic inflammation and persistent oxidative stress)chronic inflammation and persistent oxidative stress) and generally anticipate, to some extent preparing it, genetic modification and an overall genomic instability

• … could these data change our way of representing cancer ?

** and hypermethylation of tumor suppressor genes promoters

towards an epigenetic paradigm towards an epigenetic paradigm

Cancer cells present a gain of methylated streches at regions that are usually unmethylated (hypermethylation) concomitantly withloss of methylation at genomic loci that are normally methylated (global) (hypomethylation),

The ‘‘methylation paradox’’ of cancer cells.

Retrosequences activation

R Villa, F De Santis, A Gutierrez, S Minucci, PG Pelicci, L Di Croce Epigenetic gene silencing in acute promyelocytic leukemia Biochem Pharmacol (2004) 68: 1247-54

Interphase chromosomeMitotic chromosome

Euchromatin

Heterochromatin

What’s Epigenetics ?What’s Epigenetics ?

FOREWORD 2 FOREWORD 2

In 1997 the well known molecular biologist R. Strohman attempted an oblique attackattack against the against the central dogma central dogma of of molecular biologymolecular biology; the deterministic, linear, uni-directional, and encapsulated path from DNA to RNA to proteins to phenotype..

We have wrongly extended the linear theory of the gene to the “realm” of the gene management... but the gene management is an entirely different process, involving interactive interactive cellular cellular processes that processes that display an display an interactive interactive complexity… complexity… which is which is epigenetic in epigenetic in naturenature1

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Francis Crick's statement of the central dogma, from an early draft of Crick (1958)available at http://profiles.nlm.nih.gov/SC/B/B/F/T/_/scbbft.pdf

Bromham L Biol. Lett. 2009;5:503-505©2009 by The Royal Society

If the Central Dogma of Molecular Biology depicted one direction-flow of genetic information………………

REVERSETRANSCRIPTION

TRANSCRIPTIONFACTORS

(and COFACTORS)

GRNetworks

SYSTEMS GENOMICS(BIOLOGY)

NATURAL GENETIC ENGINEERING

TRANSPOSABLE ELEMENTS

The “Fluid (Epi)Genome”

EPIGENETIC CHANGES

ENVIRONMENT

we now know that things are quite different: information flow is circular between genome and environment

Shapiro JA. Revisiting the central dogma in the 21st century Ann N Y Acad Sci. (2009);1178:6-28

….Genes Know How to Network…BUT...

http://news.sciencemag.org/sciencenow/2009/04/21-03.html

IN FACT GenesIN FACT Genes need to be told to switch “off” need to be told to switch “off” and “on”:and “on”:• Genes need to be told how much expression (protein) is required and where.• Genes need to be regulated Genes need to be regulated – this regulation regulation is not performed by DNAis not performed by DNA but by many other but by many other controls arranged in a controls arranged in a complex networkcomplex network• DNA has been called the Book of Life by the Human Genome Project scientists, but many other biologists consider DNA to be simply a DNA to be simply a random collection of words random collection of words from which a from which a meaningful story of life may be assembled…meaningful story of life may be assembled…• In In order to assemble that meaningful story, a living cell uses a second informational systemcell uses a second informational system. (...) The key concept here is that these these dynamic-epigenetic networks have a life of dynamic-epigenetic networks have a life of their own —they follow network-rules not their own —they follow network-rules not specified by DNAspecified by DNA

From directing the fate of stem cells to determining how.. we grow, the genes in our the genes in our body act in complex networks..body act in complex networks.. the whole Genome is a Complex and highly dynamic molecular Network of interacting Genes and non-codifying sequences.. and proteins

Strohman R. , April 2001Beyond genetic determinism

Naesens, M. & Sarwal, M. M Current paradigm of molecular biology Nat. Rev. Nephrol. 6, 614–628 (2010)

..configuring a new model of genomic molecular network composed by DNA, epigenome, proteome sequences communicating in cis and trans

and, above all, reactive /interactive (in a continuous dialogue with the environment)

Hormons

Xenobiotics

EMF

VIRUSES

TCDD

Viruses

HERVs

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SYNERGISM ?!

“FLUID EPI-GENOME”

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We may represent the environment as a continuous stream of information (simple: photons: individual packages of E = M = Information) or complex (organic molecules, viruses etc) interacting with our cells [membrane or transmembrane receptors, signal transduction proteins, nuclear receptors, genome (DNA + Epigenome)] forcing them to adapt

We may represent the environment as a continuous stream of information (simple: photons: individual packages of E = M = Information) or complex (organic molecules, viruses etc) interacting with our cells [membrane or transmembrane receptors, signal transduction proteins, nuclear receptors, genome (DNA + Epigenome)] forcing them to adapt

Rudolf Jaenisch- Whitehead Institute and Dept. of Biology, MIT, Cambridge, MARudolf Jaenisch- Whitehead Institute and Dept. of Biology, MIT, Cambridge, MA

What’s Epi-Genetics ?

The Histone tailsare a critical determinant of chromatin structure

The tails of histones could be regarded as the sensory / sensory / receptive receptive componentcomponent of the genome

Histone Tails are subject to a variety of covalent modifications

Histone Code”hypothesis: modifications of the Histone tails act as marks read by other proteins to control the expression or replication of chromosomal regions

E.g. generally, Histone Acetylation is associated with transcriptionally active genes

Deacetylation is associated with inactive genes(= gene silencing)

DNA methylation

Covalent modification of the DNA is important for gene silencing human cells. Most genes have GC rich areas of DNA in their promoter regions. These are referred to as CpG islands.Methylation of the C residues within the CpG islands leads to gene silencing

(highly unstable base)

Nuclear Receptor DNA Response Element

Histone Lysine Acetylation

Histone Deacetylases.

Histone Acetyltransferases;

Histone Methyltransferases

ATP-dependent Nucleosome Remodeling Complex

Many toxicants cause rapid alterations in gene expression by activating protein kinase signaling

cascades.

The resulting rapid, defensive alterations in

gene activity require the transmission of a signal directly to the histones

present in the chromatin of stress response genes:

within minutes of exposure

the phosphorylation of serine 10 of histone H3

and the acetylation

of lysines 9 and/or 14 take place

H3-K9 H3-S10

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The “meeting-point” between the information coming from the environment and the information encoded in the DNA (hardware) is the epigenome (software): mimetic molecules (EDCs) and other pollutants or danger-signals induce the epigenome to change

Chromatin itself is the direct target of many toxicants * … toxicant-induced perturbations in chromatin structure may precipitate adverse effects.. Forcing genome to change

Chromatin itself is the direct target of many toxicants * … toxicant-induced perturbations in chromatin structure may precipitate adverse effects.. Forcing genome to change

.. we have identified several classes of environmental chemicals that modify epigenetic marks.. including - metals (cadmium, arsenic, nickel, chromium, CH3-mercury), - peroxisome proliferators (trichloroethylene, dichloroacetic acid…), - Air Pollutants (PM 0,1/2,5/10, black carbon, benzene), - EDCs - Endocrine-Disrupting/reproductive toxicants (DES, bisphenol A, persistent organic pollutants, dioxin).

Because these epigenetic changes are small, potentially epigenetic changes are small, potentially cumulativecumulative, , and they may develop over timeand they may develop over time, it may be difficult to establish the cause-effect relationshipsdifficult to establish the cause-effect relationships among among environmental factors, environmental factors, epigenetic changesepigenetic changes, and diseases, and diseases..

….particular awareness has aroused a study that documented the presence of (geno) toxic and mutagenic substances in all the umbilical cords tested, demonstrating the ubiquity of embryo-fetal exposure

Mothers Milk: Record levels of toxic fire retardants found in American mothers' breast milk. Washington, DC. Available at http://www.ewg.org/reports/mothersmilk/NAS (National Academy of Sciences). 2000. Scientific Frontiers in Developmental Toxicology and Risk Assessment. Committee on Developmental Toxicology. Washington, DC: National Academies Press; EPA (U.S. Environmental Protection Agency). 2003a. America's children and the environment. Measures of contaminants, body burdens, and illnesses. Available online at http://www.epa.gov/envirohealth/children.

Dioxin and Dioxin-like molecules

(Ultra)-fine particles

Heavy Metals

Polycyclic Aromatic Hydrocarbons (PAH)

Benzene

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Fetal Programming

Differentiation

epi-mutations

Cellular Differentiation: an Epigentic process Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a single zygote to a complex system of tissues and 200 cell types (genetically identical.. each with its own epigenetic and morpho-functional characteristics)..

Gametogenesis. Maturation of germ cells is characterized by an impressive degree of cellular restructuring and gene regulation that involves remarkable genomic reorganization. These events are finely tuned, but are also susceptible to the introduction of various types of “error”

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Nature 447, 425-432 (24 May 2007)

Decreasing Plasticity

… although twins are epigenetically indistinguishable during the early years of life, … older monozygous older monozygous twins exhibited twins exhibited remarkable remarkable differences in differences in their overall content overall content and genomic distribution of 5-methylcytosine DNA and histone acetylation, affecting their gene-expression portrait.

3-year-old twins 50-year-old twins3-year-old twins 50-year-old twins

Epigenetic differences in homozygotic twins

Fraga et al., PNAS. Jul 26 (2005);102(30):10604-9..

Epigenetic differences arise during the Epigenetic differences arise during the lifetimelifetime of monozygotic twins

XXI e siècle: transformation spectaculaire de l'environnement et du microenvironnement utérine

Exposition Windows

The gift our mothers never wanted to give us

http://www.ewg.org/reports/generations/

ULTRAFINE PARTICLES

HEAVY METALS

ENDOCRINE DISRUPTORSENDOCRINE DISRUPTORS

The Environmental Toxic Burden and the DevelopmentalOrigins of Health and Diseases

Le fardeau de l'environnement toxique et les origines développementales de la santé et des maladies

X rays + EMFs

XENOBIOTICS

What is the Global Chemical Burden..

Industrial chemicals in mothers and daughters: the pollution we share and inherit

Is it true that these pollutants are present in blood and tissues of all men and women living in urban and industrial environments and even in the cord blood and placental and fetal tissues in more and more significant amounts year after year ?

Is it true that metals, dioxins and other lipophilic pollutants, accumulated in maternal tissue, may pass, even many years after their absorption, into the blood and reach the fetus?

Obesity/Metabolic Syndrome

Neurobehavioral Deficits and Diseases

CANCER

Reproductive Diseases/Dysfunctions

Multiorgan Effects of Endocrine Disruptors

In Vitro Fertilization

Semen Abnormalities

ObesogensDOHAD

Asthma and allergies

Psychiatric Diseases

Cardiovascular Diseases

Ipertension

Developmental Time Windows of Vulnerability

Pesticides

Lung Development

Placenta: Prediction of Future Health

Materno Fetal Stress

Cheryl Lyn Walker UT MD Anderson Cancer Center

Ce sont des quantités minimales de molécules (epi)génotoxiques, induisant des transformations continuelles de la chromatine, qui constituent le véritable problème. C’est un processus très lent pouvant démarrer lors des premières étapes du développement fœtal. Et, même dans les gamètes. Si les tissus du fœtus sont mal programmés au début et s’il y a un stress épigénétique progressif, les mutations génétiques et chromosomiques vont davantage se produire

(4) Tomatis legacy(4) Tomatis legacyRenzo TomatisRenzo Tomatis - for over 10 years Director of IARC, for nearly 20 years

Scientific Director of ISDE - had already warned us since 1979had already warned us since 1979, , on the on the Journal of the National Cancer Institute MonographJournal of the National Cancer Institute Monograph,

• that the exposure of pregnant animalsexposure of pregnant animals to chemical carcinogens can induce tumours in the offspringinduce tumours in the offspring;

• supporting his thesis with a copious scientific literature concerning 38 different (pro)carcinogenic agents;

• reminding us thatreminding us that, unfortunately, even then, , unfortunately, even then, there was there was epidemiological evidence concerning human beings, since dozens epidemiological evidence concerning human beings, since dozens of "DES daughters" had developed an adenocarcinoma of the of "DES daughters" had developed an adenocarcinoma of the vaginavagina (moreover some studies on mice and rats exposed to carcinogens during pregnancy had shown a high incidence of tumours not only in the litter of first generation, but also in the offspring of the second and third generation, providing a strong evidence of a possible transgenerational transmission of cancerpossible transgenerational transmission of cancer)

Tomatis L. Prenatal exposure to chemical carcinogens and its effect on subsequent generations. Natl Cancer Inst Monogr. (1979); (51):159-84.

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Diethylstilbestrol

MOREOVERDES Daughters, were NOT ONLY at an NOT ONLY at an increased risk for clear increased risk for clear cell cell adenocarcinomaadenocarcinoma((CCACCA) of the) of the vagina vagina andand cervix cervix, and, andbreast cancer breast cancer (2.5 fold increase in after age 40) BUT ALSO ofBUT ALSO of reproductive tract structural differences,pregnancy complications infertility and auto-immune disorders… MOREOVERsome effects of DES could be transgenerational

J Pediatr Hematol Oncol 2003; 25:635-636

The DES-tragedy could contribute to clarify the problem: endocrine-mimetic molecules and many other pollutants may act by altering the epigenetic fetal programming ..

.. DES exposure induces changes in the expression of several uterine genes involved in tissue patterning, such as Wnt7a, Hoxa9, Hoxa10, and Hoxa11, contributing to changes in tissue architecture and morphology …

.. DES exposure induces changes in the expression of several uterine genes involved in tissue patterning, such as Wnt7a, Hoxa9, Hoxa10, and Hoxa11, contributing to changes in tissue architecture and morphology …

Cancers in adults predominantly arise in (epithelial) tissues chronically exposed to environmental stress and in cells and tissues continually urged to respond/react to it

Cancers in adults predominantly arise in (epithelial) tissues chronically exposed to environmental stress and in cells and tissues continually urged to respond/react to it

While almost all childhood cancers belong to three major groups:45% oncohaematologic tumors (leukemias and lymphomas) 25% brain tumors25% neoplastic degeneration of embryonal residuals

While almost all childhood cancers belong to three major groups:45% oncohaematologic tumors (leukemias and lymphomas) 25% brain tumors25% neoplastic degeneration of embryonal residuals

The increase particularly affects children in their first life year (the incidence rate increased by> 2%)

(5) (5) Infant Infant CancerCancer• As for cancers in infantscancers in infants we should point out that

at least the first stages of the malignant processat least the first stages of the malignant process are already present at time of birth.

• The importance of genetic events in utero has been suspected, for many years, on the basis of the correlation studies on twins with leukemia.

• That some cases of leukemia originate in utero is also the result of genetic studies that have found in blood samples (Guthrie cards), taken from infants who subsequently would develop leukemias, translocations and gene sequences corresponding translocations and gene sequences corresponding to the fusion genes to the fusion genes later found in leukemic blasts.later found in leukemic blasts.

Greaves MF, Maia AT, Wiemels JL, Ford AM. Leukemia in twins: lessons in natural history Blood (2003) 1;102(7):2321-33; Mori H, Colman SM, Xiao Z, Ford AM, Healy LE, Donaldson C, et al. Chromosome translocations and covert leukemic clones are generated during normal fetal development. Proc Natl Acad Sci U S A (2002);99:8242-7

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Prenatal origin of certain childhood leukaemias

Le processus de cancérogénèse est généralement très long… Donc, le véritable problème est l’exposition des parents (gamètes) aux facteurs de risque. Le processus de cancérogénèse a débuté bien avant la naissance de l’enfant *.

Le processus de cancérogénèse est généralement très long… Donc, le véritable problème est l’exposition des parents (gamètes) aux facteurs de risque. Le processus de cancérogénèse a débuté bien avant la naissance de l’enfant *.

On peut aussi dire que la phase d’initiation * tumorale (1° Hit de Knudson) survient lors du développement ontogénétique du fœtus

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Il n’y a que deux possibilités:1) l’exposition du fœtus à des agents physiques (X-rays), chimiques ou biologiques (virus) (transmis par transmission trans-placentaire ) qui puissent endamager directement le foetus 2) la transmission trans-generationelle d’une ou plusieurs lesions genètiques ou epi-génetiques

The real question is: are these (epi) mutations stochasticor provoked by environmentally induced stress ?!?

The real question is: are these (epi) mutations stochasticor provoked by environmentally induced stress ?!?

(epi)mutations in gametes

Transgenerational Carcinogenesis

(5b) Pro-leukemic translocations in foetuses(5b) Pro-leukemic translocations in foetuses

• Moreover, pro-leukemic translocations pro-leukemic translocations are found in foetuses in foetuses with a much higher frequency than the incidence of leukemias:

• the common leukemic fusion genes: - TEL-AML1 t (12; 21) (p12; q22) and - AML1-ETO t (8; 21) (q22q22),

• are found in the cord blood with a frequency 100 times greater frequency 100 times greater than the corresponding risk of leukemiathan the corresponding risk of leukemia

Greaves M. Pre-natal origins of childhood leukaemia Rev Clin Exp Hematol. (2003);7(3):233-45

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.. the first unambiguous evidence for a prenatal origin of leukaemia was derived from studies in identical twins with leukaemia. A case of identical (monozygotic) infant twins with leukaemia was recorded in 1882, and, since that time, more than 70 pairs have been published albeit in variable detail ...

The concordance rate of leukaemia varies according to subtype and age. For infants with ALL, the rate is exceedingly high (> 50%), for “COMMON” child-ALL, is ~10%.

Adult leukaemia (ALL/ AML), in contrast, has a very low rate of concordance (< 1%).

Chromosomal translocations and preleukaemic clones arise at a substantially higher frequency (~100 X) before birth than the cumulative incidence or risk of disease, reflecting the requirement for complementary and secondary genetic events that occur postnatally. A consequence of the latter is a very variable and occasionally protracted postnatal latency of disease (1—15 years).

~1% of newborns had TEL-AML1 positive B lineage clones…which represents 100 times the incidence of TEL-AML1 positive ALL (~1 in 12,000).

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(5c) Pro-leukemic translocations as(5c) Pro-leukemic translocations as an active an active potentially positive, adaptive genomic changepotentially positive, adaptive genomic change

• This fact is usually interpreted as the evidence that translocations do not necessarily determine the onset of leukemia, which would require additional additional genetic eventsgenetic events during the postnatal period.

• An equally interesting interpretation consists in assuming that if less than 1% of children who have "produced" a translocation have developed leukemia, it could be because the translocation is an active translocation is an active potentially positive, adaptive genomic changepotentially positive, adaptive genomic change, which could be responsive to toxic exposures responsive to toxic exposures in uteroin utero

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In such a context: should TRANSLOCATIONS be considered as chromosomal aberrations or as (re)active rearrangements(re)active rearrangements ??

(6)… (6)… Pro-leukemic translocationsPro-leukemic translocations as as active/adaptive genomic changesactive/adaptive genomic changes

It may be useful to remember something very interesting occurring in areas with persisting in areas with persisting pollution, as in Seveso,pollution, as in Seveso, where

decades after the accident which caused the dioxin contamination of a wide territory,

many individuals have been showing a high number of translocations 14:18 translocations 14:18 (typical of follicular lymphoma), although without without developing lymphomadeveloping lymphoma. .

Baccarelli A, Hirt C, Pesatori AC, Consonni D, Patterson DG Jr, Bertazzi PA, Dölken G, Landi MT. t(14;18) translocations in lymphocytes of healthy dioxin-exposed individuals from Seveso, Italy Carcinogenesis (2006); 27(10):2001-7

Exposure to NHL-associated carcinogens,

such as dioxin or pesticides, may cause expansion of t(14;18)-positive clones.

6b)… 6b)… pro-leukemic translocationspro-leukemic translocations as as active/adaptive genomic changesactive/adaptive genomic changes

It is important to note that the same translocation is frequent in subjects long exposed to pesticides for professional reasons, which is a clear evidence that

it is not the single chemical agent not the single chemical agent to determine to determine the specificity of the mutationthe specificity of the mutation, but rather, the active reaction of the genome itselfthe active reaction of the genome itself. .

Agopian J, Navarro JM, Gac AC, Lecluse Y, Briand M, Grenot P, Gauduchon P, Ruminy P, Lebailly P, Nadel B, Roulland S. Agricultural pesticide exposure and the molecular connection to lymphomagenesis J Exp Med. (2009) 6;206(7):1473-83

Figure 2. t(14;18)+ cells in HI are actively transcribing BCL2 from the translocated allele

Agopian et al. Journal of Experimental Medicine 2009:206:1473-1483

We can find exactly the same (reactive) translocation (++ expression of the anti-apoptotic gene BCL-2)in many subjects chronically exposed to pesticides ..

We can find exactly the same (reactive) translocation (++ expression of the anti-apoptotic gene BCL-2)in many subjects chronically exposed to pesticides ..

IN THE CANCEROUS B CELLS, THE PORTION OF CHROMOSOME 18 CONTAINING THE BCL-2 LOCUS HAS UNDERGONE A RECIPROCAL TRANSLOCATION WITH THE PORTION OF CHROMOSOME 14 CONTAINING THE ANTIBODY HEAVY CHAIN LOCUS. THIS T(14;18) TRANSLOCATION PLACES THE THIS T(14;18) TRANSLOCATION PLACES THE BCL-2BCL-2 GENE CLOSE TO THE HEAVY CHAIN GENE GENE CLOSE TO THE HEAVY CHAIN GENE ENHANCERENHANCER. .

H Chain-enhancer is very active in B cells...

t(14;18)(q32;q21) (IgH/IgH/BCL2BCL2)If we remember that in this, as in other pro-leukemic

or pro-lymphoma translocations, an oncogene (BCL-2: an anti-apoptotic geneBCL-2: an anti-apoptotic gene, physiologically , physiologically activated in memory lymphocytes populations and activated in memory lymphocytes populations and in in B lymphocytes "immortalized" by Epstein Barr virusB lymphocytes "immortalized" by Epstein Barr virus)) is is constantly activatedconstantly activated

because of the juxtaposed juxtaposed enhancer sequence of the enhancer sequence of the gene coding for the immunoglobulin heavy chaingene coding for the immunoglobulin heavy chain,

the hypothesis that these translocations are genomic genomic active, adaptive (potentially defensive) changesactive, adaptive (potentially defensive) changes rather than simple "chromosomal aberrations" rather than simple "chromosomal aberrations" acquires a meaning and a greater value …

Kurtulus S, Tripathi P, Moreno-Fernandez ME, Sholl A, Katz JD, Grimes HL, Hildeman DA.Bcl-2 allows effector and memory CD8+ T cells to tolerate higher expression of Bim J Immunol.(2011) 15;186(10):5729-37; Finke J, Fritzen R, Ternes P, Trivedi P, Bross KJ, Lange W, Mertelsmann R, Dölken G.Expression of bcl-2 in Burkitt's lymphoma cell lines: induction by latent Epstein-Barr virus genes Blood. (1992); 15;80 (2):459-69

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(7) InfantInfant leukemias

In conclusion, we must remember that infant leukemiasinfant leukemias

– which, as mentioned, have registered the highest increase and, according to this dynamic model, may represent the most emblematic the most emblematic consequence of a distorted consequence of a distorted fetal programmingfetal programming (an “evolutionary process gone awry”) -

show, even at a even at a molecular levelmolecular level, some , some peculiar peculiar characteristicscharacteristics

Soto AM, Maffini MV, Sonnenschein C. Neoplasia as development gone awry: the role of endocrine disruptors. Int J Androl.(2008);31(2):288-93

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(7b) MLL1 As a matter of fact it is significant that in these forms

the MLL1 geneMLL1 gene - a gene encoding aencoding a histone methyltransferasehistone methyltransferase - an enzyme

playing a key role in the implementation of epigenetic and chromatin modifications which are fundamental in the early stages of fetal development and differentiation of tissues (and especially in emo-lymphopoiesis) -

- is very often involved and implicated in dozens of different in dozens of different translocations that express fusion proteins translocations that express fusion proteins capable of interfering with the differentiation of pluripotent hematopoietic stem cells, dysregulating the expression dysregulating the expression patterns of HOX genespatterns of HOX genes

Ernst P, Wang J, Korsmeyer SJ. The role of MLL in hematopoiesis and leukaemia Curr Opin Hematol.(2002); 9(4):282-7; Popovic R, Zeleznik-Le NJ. MLL: how complex does it get? J Cell Biochem. (2005) 15;95(2):234-42; De Braekeleer M, Morel F, Le Bris MJ, Herry A, Douet-Guilbert N. The MLL gene and translocations involving chromosomal band 11q23 in acute leukaemia Anticancer Res. (2005); 25(3B):1931-44

Nakamura T, Mori T, Tada S, et al. ALL-1 is a histone methyltransferase that assembles a supercomplex of proteins involved in transcriptional regulation. Mol Cell 2002;10:1119-1128.

IN ALL AND AML, THE ALL1 (ALSO NAMED MLL) GENE CAN FUSE WITH 1 OF MORE THAN 50 GENES. ALL1 IS PART OF A MULTIPROTEIN

COMPLEX. MOST OF THE PROTEINS IN THE COMPLEX ARE COMPONENTS OF TRANSCRIPTION COMPLEXES; OTHERS ARE INVOLVED IN HISTONE

METHYLATION AND RNA PROCESSING. THE ENTIRE COMPLEX REMODELS,

ACETYLATES, DEACETYLATES, AND METHYLATES NUCLEOSOMES AND HISTONES. THE FUSION OF ALL1 WITH 1 OF these 50 PROTEINS RESULTS IN THE FORMATION OF THE CHIMERIC PROTEINS THAT UNDERLIE ALL AND AML.

ALL1 (MLL) FUSION PROTEINS DEREGULATE HOMEOBOX GENES (WHICH

ENCODE TRANSCRIPTIONS FACTORS)..and microRNAs GENES SUCH AS MIR191.

AF9 Location 9p22

MLL Location 11q23

Several lines of evidence point to a mishap in non-homologous end joining of double strand breaks as the most likely reason for 11q23 translocations.

The first and most striking property of MLL fusion proteins is their incredible diversity. MLL has been found in 73 different translocations and 54 partner genes have been cloned (http://atlasgeneticsoncology.org/Genes/MLL.html).

histone methyltransferasehistone methyltransferase

Translocations typical of myeloid leukaemia, probably due to maternal exposure to some toxic compound, were shown to be present at birth in children who developed the disease years later (while not sufficient per se to cause the disease, they might increase the risk for leukaemia by inducing genomic instability) Tomatis L. Identification of carcinogenic agents and primary prevention of cancer. Ann N Y Acad Sci. 2006 Sep;1076:1-14

Translocation involving band 11q23 in AML may occur as a result of a deletion or trans-locations with a number of other chromosomes and is usually associatedwith M4 or M5 and a poor prognosis

Even if leukaemia fusion gene leukaemia fusion gene formation isformation is spontaneous, the risk of this occurring may be modified by other factors, including folate availability. There is dietary and genetic evidence that folate has an impact on the risk of infant and childhood leukaemia ..

MLL rearranged leukemias are associated with poor prognosis and very brief latency for MLL-AF4+ infant B ALL. This raises the question of how this disease can evolve so quickly,!

!!

MLL1-gene reactivereactive rearrangements• Even the high frequency of the high frequency of MLL1-gene rearrangementsMLL1-gene rearrangements in in

leukemias and myelodysplastic syndromes leukemias and myelodysplastic syndromes secondary to secondary to treatment with topoisomerase II inhibitorstreatment with topoisomerase II inhibitors is significant is significant as an argument in favour of the prenatal and (epi)geno-as an argument in favour of the prenatal and (epi)geno-toxic origin of infant leukemia, toxic origin of infant leukemia,

• due to maternal and fetal exposure to substances maternal and fetal exposure to substances capable of interfering with the action of this enzymecapable of interfering with the action of this enzyme, which is essential for the unwinding of the double helix, such as such as bioflavonoidsbioflavonoids (contained in many foods) and (contained in many foods) and widely used widely used insecticides such as insecticides such as dipyrone (Baygon)dipyrone (Baygon)

Strick R, Strissel PL, Borgers S, Smith SL and Rowley JD: Dietary bioflavonoids induce cleavage in the MLL gene and may contribute to infant leukemia. Proc Natl Acad Sci USA (2000) 97: 4790-4795 ; Alexander FE, Patheal SL, Biondi A, Brandalise S, Cabrera ME, Chan LC, Chen Z, Cimino G, Cordoba JC, Gu LJ, Hussein H, Ishii E, Kamel AM, Labra S, Magalhães IQ, Mizutani S, Petridou E, de Oliveira MP, Yuen P, Wiemels JL, Greaves MF. Transplacental chemical exposure and risk of infant leukemia with MLL gene fusion Cancer Res. (2001) 15; 61(6):2542-6

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Our study has supported the hypothesis that in utero exposure to chemicals causes MLL* infant leukemia and has generated specific hypotheses that require further testing. Exposure to dipyrone is widespread, particularly in Central and South America where it is available as an inexpensive, nonprescription drug. Mosquitocidals are similarly in general use in these same settings. Propoxur (Baygon°) is also widely used against cockroaches, fleas, and similar pests. Therefore, it is important that the associations observed in this study are reevaluated in an extended case-control study

This is the first demonstration that transplacental benzene exposure can induce hepatic and hematopoietic tumorsin mice, which may be dependent on fetal benzene metabolism capability

REMARK

• Those who adhere to the paradigm of paradigm of stochastic mutationsstochastic mutations

• and more generally to a linear and gene-to a linear and gene-centric model of DNA centric model of DNA • have obviously some difficulty to obviously some difficulty to

accept all this…accept all this… 8888

The dynamic (epi)-genomeThe dynamic (epi)-genomeIn fact oonly in a model of genome conceived as nly in a model of genome conceived as

a a unitaryunitary and and complexcomplex (at the same time, (at the same time, dynamicdynamic and and responsiveresponsive) ) molecular networkmolecular network,,

it is possible to suggest that all the epigeneticepigenetic (global global DNA hypomethylation, hyper-methylation of promoter DNA hypomethylation, hyper-methylation of promoter sequences of tumor suppressor genes..sequences of tumor suppressor genes..), geneticgenetic (genomic instability, mobilization of transposable genomic instability, mobilization of transposable sequences..sequences..), and chromosomalchromosomal (translocationstranslocations)

mutationsmutations, determining the progression of cancer,should be seen as should be seen as steps in a (failed or distorted) steps in a (failed or distorted)

evolutionary/adaptive and defensive processevolutionary/adaptive and defensive process

Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps Nat Rev Genet (2007);8(4):286-98; Karpinets TV, Foy BD. Tumorigenesis: the adaptation of mammalian cells to sustained stress environment by epigenetic alterations and succeeding matched mutations. Carcinogenesis. (2005); 26(8):1323-34; Hauptmann S., Schmitt W.D. Transposable elements - Is there a link between evolution and cancer? Medical Hypotheses (2006), 66 (3):580-591;

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In pre-neoplastic lesions pre-neoplastic lesions we find some common we find some common epigenetic changesepigenetic changes, produced by , produced by prolonged prolonged stressstress: : an hypo-methylation of the whole DNA sequence, a hyper-methylation of the promoters of onco-suppressor genes, a global genomic instabilityIn pre-neoplastic lesions pre-neoplastic lesions we find some common we find some common epigenetic changesepigenetic changes, produced by , produced by prolonged prolonged stressstress: : an hypo-methylation of the whole DNA sequence, a hyper-methylation of the promoters of onco-suppressor genes, a global genomic instability

These mechanisms may be activated in cells by continuing proliferative and survival signaling in a sustained stressenvironment (SSE)… longterm exposure to this signaling epigenetically reprograms the genome of some cells..

Mutations match epigenetic tags

Selection of Mutants follows !

Many epigenetic tags are Induced in stem cells during fetal programming

tags = marcature

Transposable elements Transposable elements can be seen as a can be seen as a natural genetic engineering systemnatural genetic engineering system capable of acting capable of acting on the genome as a whole on the genome as a whole .. This dynamic view of the genome has been illustrated most impressively by Shapiro who stated that the genome is composed of modular units arranged the genome is composed of modular units arranged in a “Lego-like” manner in a “Lego-like” manner that can be altered under certain circumstancesthat can be altered under certain circumstances

The hypothesis developed in this article is that chronic chronic

stressors can induce stressors can induce adaptive rearrangements adaptive rearrangements

of the genome of the genome which might result in new proteins

helping survival in a harmful environment or trigger the

development of a completely ‘‘new’’ organ –

the cancer – which also survives – even though its

survival is at the expenses of its host.

Stressors, which cannot be compensated for with the usual cell possibilities might arouse evolutionary mechanisms intended to create new protein variants. One of these is the One of these is the activation of transposable elements activation of transposable elements which leads to a reformatting of the genomewhich leads to a reformatting of the genome.

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Our study demonstrates the strong link strong link betweenbetweenhypomethylation of hypomethylation of transposable elements transposable elements with with genomic instabilitygenomic instabilityin non-small cell lung cancer and provides early evidence for early evidence for a potential active role a potential active role of these elements of these elements in lung neoplasia.in lung neoplasia.

Retrosequences activation…

MOLECULAR WEIGHT OF DIFFERENT PROTEINS PRESENT IN ZEBRAFISH EMBRYO DURING GASTRULATION PERIOD

1) 37% molecular weight of about 97KDa2) 14.6% molecular weight of about 45 KDa3+4) 27.4% molecular weight of about 30-25 KDa5) 4% molecular weight of about 20 KDa6+7) 14% molecular weight of about 14 KDa

Biava M. et al Embryonic morphogenetic field induces phenotypic reversion in cancer cells. Current Pharmaceutical Biotechnology Volume 12, Issue 2, 2011, Pages 243-253

..placing tumor cells into a "normal" morphogenetic field - like that of an embryonic tissue - one can reverse malignant phenotype, "reprogramming" tumor into normal cells.

Genes(Fluid) Genome

Phenotype

Phenotype

Random Mutations

Neodarwinistic Paradigm

Neo-Lamarckian-ConstructiveParadigm

Natural Selection

Natural Selection

Environment

(Fluid) Epigenome

Proteome

(Environment)

(microevolution)

microevolution

Macroevolution

RibotypeReverese

Transcriptase

Mobile Sequences

Symbiogenesis

Horizontal gene transfer

(ecosystems)Natural Genetic Engineering

Rs: Receptors

Cells Rs

Molecules

Organisms

HATs

HDACs

NRs

DMTs

TFs

Rs

GRNsMaster Genes

A

B

EMERGENT

PROPERTIES Environmentalcrisis

NEW DEVELOPMENTAL PATTERNS

Conclusions (a)This was just a brief introduction to an extremely complex

and delicate issue, concerning the meaning to be given to the increase in childhood cancers, in recent decades,

together with the (even more pronounced) increase and the together with the (even more pronounced) increase and the progressive anticipation of the age of the onset of many progressive anticipation of the age of the onset of many chronic degenerative and inflammatory diseaseschronic degenerative and inflammatory diseases.

It is not possible so far to prove with absolute certainty the embryo-fetal (epigenetic) origins of these diseases

But decades after the first "decades after the first "propheticprophetic" analysis made " analysis made by by Renzo Tomatis Renzo Tomatis

and the first explicit formulation of a theory about the foetal origin of some cancers

D. Trichopoulos Hypothesis: does breast cancer originate in utero? Lancet (1990), 335, pp. 939–940; Sanderson ML et al. Perinatal factors and risk of breast cancer Epidemiology (1996) 7, 34-37; Michels KB Birthweight as a risk factor for breast cancer Lancet, (1996)348, 1542–1546; Vatten L Can prenatal factors influence future breast cancer risk? The Lancet, (1996) 348, 9041, 1531

Conclusions (b) … in a world increasingly characterized by world increasingly characterized by ubiquitous distribution of ubiquitous distribution of

thousands of potentially pro-carcinogenic molecules in food thousands of potentially pro-carcinogenic molecules in food chains and even in the cord bloodchains and even in the cord blood

• it is necessary to recall, once again, the great lesson of Tomatis, who claimed that in order to reverse the trend of the continuous increase in tumors there is an obligatory path to take, that of the primary prevention id est a reduction in the overall chemical burden, concerning

• not only those substances for which there is sufficient evidencenot only those substances for which there is sufficient evidence in experimental studies of chemical carcinogenesis,

• but alsobut also for the numerous "possible" carcinogens possible" carcinogens ((IARC group 2BIARC group 2B),), for many of which it is highly for many of which it is highly unlikely that any future research will unlikely that any future research will give a definitive answergive a definitive answer about their carcinogenicity

Christiani DC Combating Environmental Causes of Cancer N Engl J Med. (2011) 3; 364(9):791-3

Tomatis L. Role of experimental and epidemiological evidence of carcinogenicity in the primary prevention of cancer. Ann Ist Super Sanita. (2006);42(2):113-7; Tomatis L. Identification of carcinogenic agents and primary prevention of cancer Ann NY Acad Sci. (2006);1076, 1-14

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Fetal programming

1

53

2

Ontogenesis

Phylogenesis

Developmental Plasticity

Devo-Evo

4 Mismatch 6

7 XX Century Epidemiologic

TransitionEnvironment

From Genetics to Epigenetics

The 7 keys