Genetic Basis of Cancer: the Origins

• Circa 1860: microscopist Mueller discovers that

tumor is made of cells
• 1908: Ellerman and Bang demonstrate that a
filterable agent induces leukemia in chicken
• 1911: Rous demonstrates that a filterable agent
induces sarcomas in chicken
• 1936: Bittner that mammary cancer can be
transmitted through milk in a specific mouse strain
• 1951: Gross discovered the first murine leukemia
virus
The SV40 oncogenic
polyomavirus
Retrovirus vital cycle
The largest part
of retroviral
oncogenes are
tyrosin kinase
proteins (src, fps,
yes, fgr, ros, abl,
erbB, kit, sea)

Other oncogenes
functions as
transcription
factors (myc,
myb, fos, jun,
erbA)
 Point Mutation Activated-RAS
Oncoprotein is Required to Maintain the
Malignant Phenotype
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
 Il cromosoma Ph’ nella
Leucemia Mieloide Cronica:
La scoperta del cromosoma Ph’ nella Leucemia
Mieloide Cronica: più del 95% dei casi presenta
questa aberrazione cromosomica

Peter Nowell, Università di Pennsylvania, Philadelphia

Il cromosoma Ph’ : una traslocazione
reciproca tra il cromosoma 9 ed il 22

 
   
                                     

Janet Rowley, Università di Chicago

 Scoperta del difetto genetico:
la traslocazione t(9;22) causa la fusione dei
geni BCR-ABL
Eli Canaani, Israel

Il proto-oncogene ABL, una tirosin-chinasi,

diviene attivata dalla fusione con BCR: la sua
attivita` chinasica diviene costitutivamente
Carlo M. Croce, Italia-USA attiva a livelli elevati
Translocation
t(8;14)(q24;q32) in
Burkitt’s lymphoma
MYC is activated by juxtaposition to the Ig locus
Nonrandom chromosomal aberrations
in myeloid malignancies
Multiple translocation partners for
MLL and TEL
Nonrandom chromosomal aberrations
in lymphoid malignancies
DNA amplification leads to
oncogene over-expression
Proto-oncogenes can be activated by a
variety of mechanisms
Point mutations, leading to aberrant biochemical function

Fusion with other genes, leading to aberrant biochemical

function

Abnormally elevated level due to promoter/enhancer activation

or to gene amplification
Il fenotipo tumorigenico è
recessivo a livello cellulare

FUSIONE

Cellula Cellula
Tumorale Non Tumorale

Ibrido non tumorigenico

 TUMOR SUPPRESSOR GENES AND CANCER

Mutation 2 X
Mutation 1 X X
X

Normal Heterozygous Loss of

Heterozygosity

Other: Gene Methylation

Expression Levels
Knudson’s Two-Hits Hypothesis
Mechanisms for RB1 tumor suppressor
gene inactivation
DNA polymorphisms and loss of
heterozygosity in cancer
 DNA Methylation in
Mammalian Cells
CpG methylation is the prototype of a mammalian epigenetic mark

• In mammalian cells, cytosines present in

the 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 of CpG island
promoters and gene regulation
DNA methylation in development
 DNA methylation at replication
maintains a stable epigenotype

Inheritance of cytosine methylation:

DNA
1. the symmetrical nature of the replication

modified sequences (CpG)

2. The preference of the
“maintenance” DNMT1 for
hemimethylated DNA
DNA
Methylation
(DNMT1)
Methylation
of CpG Islands
in Gene Promoters
Represses
Gene Expression
Tumor suppressor gene inactivation
by promoter methylation
 Examples of tumor suppressor genes
inactivated by promoter methylation
Mechanisms of Inactivation Double Hit Mechanisms
Homozygous Mutation Methylation Mutation + Biallelic
LOH Mutation Methylation deletion + LOH + LOH Methylation 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
 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
microRNAs are expressed as longer precursor RNAs

Moss (2002) Current Biology 12, R138

 siRNA e miRNA, per la loro
struttura, biogenesi e meccanismo
miRNA gene
di funzionamento potrebbero
rappresentare variazioni dello
Pri-miRNA stesso processo evolutivo
Drosha
Exportin-5 Da trascrizione elementi ripetitivi e trasposoni,
RNA bicatenario
Pre-miRNA
Dicer
siRNA
miRNA Duplex
RISC

Helicase Taglio mRNA

Alcuni miRNAs
Repressione Traduzione
RISC

RISC RISC

Regione
codificante 3’ non codificante
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 miRNA registry
| | | | | +-- Homo sapiens (227) http://microrna.sanger.ac.uk/sequences/
| | | | | +-- Mus musculus (230)
| | | | | +-- Rattus norvegicus (191)
| | | | | +-- Canis familiaris (6)
| | | +-- Amphibia
| | | | | +-- Xenopus laevis (7)
| | | +-- Pisces
| | | | | +-- Danio rerio (33)
+-- Viruses
| +-- Epstein Barr virus (5)
| +-- Viridiplantae
| +-- Kaposi sarcoma-associated herpesvirus (11)
| | | | +-- Arabidopsis thaliana (114)
| +-- Human cytomegalovirus (9)
| | | | +-- Oryza sativa (173)
| +-- Mouse gammaherpesvirus 68 (9)
| | | | +-- Sorghum bicolor (64)
| | | | +-- Zea mays (40)
mir-15 and mir-16 are within the minimal
region of 13q deletion in human CLL
 Per Bl Leuk
Sk muscle
Pancreas
Prostate

Testicle
Kidney

CD5 +
Liver

BM
Expression of
70bp
mir-15 and mir-16 miR16

in normal tissues 20bp

and human CLLs 70bp
miR15

20bp
CD5 +

CLL 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
13qLOH +/+ ND +/- +/- +/- ND +/- NI +/+ +/+ NI +/- NI NI +/- +/- +/- NI

70bp
miR16
20bp

70bp
miR15
20bp
BCL2 is target of mir-15/16
BCL2 is target of mir-15/16
 Saggi luciferasici per la conferma di
bersagli genici di miRNA deregolati in
cancro microRNA

Luciferasi
3’UTR gene bersaglio

EFFETTO: RIDUZIONE ATTIVITA’ LUCIFERASICA

Effetto di mir-15a / mir-16-1 su BCL2

Let-7 down-regulates human RAS

Johnson et al. Cell 120: 635, 2005

Let-7 down-regulate human RAS

Johnson et al. Cell 120: 635, 2005

Let-7 is down-regulated in human cancer,
resulting in up-regulation of RAS protein

Johnson et al. Cell 120: 635, 2005

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

Target mRNA

n
6a io
-2 let
overexpression

?
iR e
m sd
miR transcript

6, u
/1 g o
miR promoter

15 y
iR o z
miR gene

r
m om ot e
m
H

+ Pro n
t i o n
y l at i o
le th
De erme 5/16?
hyp miR1 tion Proliferation
n + Mu t a
tio
Dele Apoptosis
Am
Invasion
plif
(mi icatio
R n
155 Angiogenesis
Tra

)
nsl
o c at
i on

R
mi
Target mRNA
O downregulation
ge ther
ne Specific effects
miR-142
promoter c-myc mRNA