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Genetic Basis of Cancer: the Origins
Circa 1860: microscopist Mueller discovers thattumor is made of cells
1908: Ellerman and Bang demonstrate that afilterable agent induces leukemia in chicken
1911: Rous demonstrates that a filterable agentinduces sarcomas in chicken
1936: Bittner that mammary cancer can betransmitted through milk in a specific mouse strain
1951: Gross discovered the first murine leukemiavirus
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The SV40 oncogenic
polyomavirus
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Retrovirus vital cycle
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The largest part
of retroviral
oncogenes aretyrosin kinase
proteins (src, fps,
yes, fgr, ros, abl,
erbB, kit, sea)
Other oncogenes
functions as
transcription
factors (myc,
myb, fos, jun,
erbA)
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HRAS was the first proto-oncogene found activated by point mutations in
human tumor cells: about 20% of all human tumors carry a mutated RAS
Its oncogenic function was demonstrated by the finding that the mutant RAS
can transform NIH3T3
Activated RAS cooperates with other oncogenes in tranforming primary
human cells.
Its activity is essential to maintain the transformed phenotype
Point Mutation Activated-RAS
Oncoprotein is Required to Maintain the
Malignant Phenotype
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Il cromosoma Ph nella
Leucemia Mieloide Cronica:
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La scoperta del cromosoma Ph nella Leucemia
Mieloide Cronica: pi del 95% dei casi presenta
questa aberrazione cromosomica
Peter Nowell, Universit di Pennsylvania, Philadelphia
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Il cromosoma Ph : una traslocazione
reciproca tra il cromosoma 9 ed il 22
Janet Rowley, Universit di Chicago
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Scoperta del difetto genetico:
la traslocazione t(9;22) causa la fusione dei
geni BCR-ABLEli Canaani, Israel
Carlo M. Croce, Italia-USA
Il proto-oncogene ABL, una tirosin-chinasi,
diviene attivata dalla fusione con BCR: la sua
attivita` chinasica diviene costitutivamente
attiva a livelli elevati
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Translocation
t(8;14)(q24;q32) in
Burkitts lymphoma
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MYC is activated by juxtaposition to the Ig locus
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Nonrandom chromosomal aberrations
in myeloid malignancies
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Multiple translocation partners for
MLL and TEL
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Nonrandom chromosomal aberrations
in lymphoid malignancies
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DNA amplification leads to
oncogene over-expression
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Proto-oncogenes can be activated by a
variety of mechanisms
Point mutations, leading to aberrant biochemical function
Fusion with other genes, leading to aberrant biochemicalfunction
Abnormally elevated level due to promoter/enhancer activation
or to gene amplification
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Il fenotipo tumorigenico
recessivo a livello cellulare
FUSIONE
Cellula
Tumorale
Cellula
Non Tumorale
Ibrido non tumorigenico
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TUMOR SUPPRESSOR GENES AND CANCER
X
X
X XMutation 1
Mutation 2
Heterozygous Loss of
Heterozygosity
Normal
Other: Gene Methylation
Expression Levels
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Knudsons Two-Hits Hypothesis
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Mechanisms for RB1 tumor suppressor
gene inactivation
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DNA polymorphisms and loss of
heterozygosity in cancer
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In mammalian cells, cytosines present inthe dinucleotide CpG are generally
methylated
CpG islands are regions 500-2000
nucleotides long enriched in CpG
dinucleotides that are found in about 60%of gene promoters.
CpG islands are usually not methylated
DNA Methylation in
Mammalian CellsCpG methylation is the prototype of a mammalian epigenetic mark
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DNA methylation of CpG island
promoters and gene regulation
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DNA methylation in development
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DNA methylation at replication
maintains a stable epigenotype
DNA
replication
DNA
Methylation
(DNMT1)
Inheritance of cytosine methylation:
1. the symmetrical nature of the
modified sequences (CpG)
2. The preference of the
maintenance DNMT1 for
hemimethylated DNA
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Methylation
ofCpG Islands
in Gene PromotersRepresses
Gene Expression
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Tumor suppressor gene inactivation
by promoter methylation
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LOH Mutation Methylation
Homozygous
deletion
Mutation
+ LOH
Methylation
+ LOH
Mutation +
Methylation
Biallelic
Methylation
RB1 Retinoblastoma yes yes yes rare yes yes yes ?
VHL Renal Ca yes yes yes rare yes yes yes ?
BRCA1 Breast/Ovarian Ca yes yes yes no yes ? ? yes
CDKN2A Melanoma yes yes yes yes yes yes yes yes
MLH1 Colorectal Ca yes yes yes ? yes ? yes yes
APC Colorectal Ca yes yes yes ? yes ? ? ?
MGMT Lung ca yes no yes no no ? no yes
DAP Lung ca yes no yes no no ? no yes
Double Hit MechanismsMechanisms of Inactivation
Examples of tumor suppressor genes
inactivated by promoter methylation
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Tumor suppressor genes are
inactivated by a variety of mechanisms
Point mutations, leading to aberrant biochemical function
Deletion leading to loss of gene
Aberrant DNA methylation leading to loss of transcriptional
activity
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Moss (2002) Current Biology 12, R138
microRNAs are expressed as longer precursor RNAs
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RISCRepressione TraduzioneRepressione Traduzione
Alcuni miRNAs
Pre-miRNAPre-miRNA
DroshaDrosha
miRNA genemiRNA gene
Pri-miRNAPri-miRNA
DicerDicer
DuplexmiRNAmiRNA
Exportin-5Exportin-5
Helicase
Regione
codificante
RISC RISC
3 non codificante
Taglio mRNATaglio mRNA
RISC
RNA bicatenarioRNA bicatenario
siRNAsiRNA
siRNA e miRNA, per la loro
struttura, biogenesi e meccanismo
di funzionamento potrebbero
rappresentare variazioni dellostesso processo evolutivo
Da trascrizione elementi ripetitivi e trasposoniDa trascrizione elementi ripetitivi e trasposoni,
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Quanti sono i miRNA attualmente noti ?
Nel registro dei microRNA, ad aprile 2005 vi erano| +-- Metazoa
| | | +-- Arthropoda| | | | | +-- Drosophila melanogaster (78)
| | | | | +-- Drosophila pseudoobscura (73)
| | | | | +-- Anopheles gambiae (38)
| | | | | +-- Apis mellifera (25)
| | | +-- Nematoda
| | | | | +-- Caenorhabditis elegans (116)
| | | | | +-- Caenorhabditis briggsae (79)
| | | +-- Vertebrata
| | | +-- Aves
| | | | | +-- Gallus gallus (122)
| | | +-- Mammalia
| | | | | +-- Homo sapiens (227)
| | | | | +-- Mus musculus (230)
| | | | | +-- Rattus norvegicus (191)
| | | | | +-- Canis familiaris (6)
| | | +-- Amphibia
| | | | | +-- Xenopus laevis (7)| | | +-- Pisces
| | | | | +-- Danio rerio (33)
| +-- Viridiplantae
| | | | +-- Arabidopsis thaliana (114)
| | | | +-- Oryza sativa (173)
| | | | +-- Sorghum bicolor (64)
| | | | +-- Zea mays (40)
+-- Viruses
| +-- Epstein Barr virus (5)
| +-- Kaposi sarcoma-associated herpesvirus (11)
| +-- Human cytomegalovirus (9)
| +-- Mouse gammaherpesvirus 68 (9)
miRNAregistry
http://microrna.sanger.ac.uk/sequences/
i 15 d i 16 ithi th i i l
http://microrna.sanger.ac.uk/sequences/index.shtmlhttp://microrna.sanger.ac.uk/sequences/index.shtmlhttp://microrna.sanger.ac.uk/sequences/index.shtmlhttp://microrna.sanger.ac.uk/sequences/index.shtml8/8/2019 2.1 Molecular Biology Cancer
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mir-15 and mir-16 are within the minimal
region of 13q deletion in human CLL
k
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BM
PerBlLeu
k
CD5+
Kidney
Prostate
Liver
Pancreas
Skmuscle
Testicle
miR16
miR15
70bp
20bp
20bp
70bp
CLL
13qLOH
1
+/+
2
ND
3
+/-
4
+/-
5
+/-
6
ND
7
+/-
8
NI
9
+/+
10
+/+
11
NI
12
+/-
13
NI
14
NI
15
+/-
16
+/-
17
+/-
18
NI
miR16
miR15
70bp
20bp
20bp
70bp
CD5+
Expression of
mir-15 and mir-16in normal tissues
and human CLLs
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BCL2 is target of mir-15/16
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BCL2 is target of mir-15/16
Saggi luciferasici per la conferma di
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Saggi luciferasici per la conferma di
bersagli genici di miRNA deregolati in
cancro
Luciferasi
3UTR gene bersaglio
microRNA
EFFETTO: RIDUZIONE ATTIVITA LUCIFERASICA
Effetto di mir-15a / mir-16-1 su BCL2
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Let-7 down-regulates human RAS
Johnson et al. Cell 120: 635, 2005
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Let-7 down-regulate human RAS
Johnson et al. Cell 120: 635, 2005
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Let-7 is down-regulated in human cancer,
resulting in up-regulation of RAS protein
Johnson et al. Cell 120: 635, 2005
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Let-7 is down-regulated in human cancer,
resultin in up-regulation of RAS protein
Johnson et al. Cell 120: 635, 2005
Main mechanism of miRNA involvement in cancer is aberrant expression
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miRprom
ote
r
miRgene
miRtranscr
ipt
Specific effects
Apoptosis
Proliferation
Invasion
Angiogenesis
Target mRNA
overexpressionoverexpression
Target mRNA
downregulation
Delet
ion+Pr
omote
rHo
mozy
gous
deletio
n
miR
15/16,m
iR-26a?
Deleti
on+Mu
tation
Translo
cation
miR
Amplification(miR155)
miR 142
hyper
methy
lation
miR1
5/16?
Othergene
p
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