Download lezioni retro2

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Gli oncogeni
Gli oncogeni dei retrovirus sono geni cellulari catturati come RNA durante la
infezione virale
Il gene virale ha accumulato delle mutazioni che lo rendono più attivo,
non regolato,
attivo intessuti diversi
gli hanno conferito nuove funzioni ( gain of function)
V-Src sarcoma in pollo: 12 aa al C-terminale hanno sostituito i 19 in c-src e
rendono la proteina attiva costitutivamente
H-ras (Rat Harvey sarcoma): 3/189 sostituzioni deregolano la proteina
V-mos (Moloney murine sarcoma): 11/369 sostituzioni rendono la proteina
(Ser chinasi di cicline) attiva costitutivamente
Molte oncoproteine sono fattori di trascrizione (Fos, Myc)
La procedura della cromatin immunoprecipitation ChIP
Servirà a definire tutti I promotori target
Crosslinked DNA+proteins
IP anti transcription factor
DNA extraction
PCR amplification and sequence
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C-FOS Feline OSteosarcoma
It was first discovered in rat fibroblasts as the
transforming gene of the FBJ MSV (Finkel–Biskis–
Jinkins Murine osteogenic Sarcoma virus).
C-fos encodes a 62 kDa protein, which forms
heterodimer with c-jun (part of Jun family of
transcription factors), resulting in the formation of
AP-1 (Activator Protein-1)
AP-1
Regola le cicline D1
Matsushime et al.
Cell 65, 1991
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AP-1 modulates cell proliferation through its ability to regulate the expression and
function of cell-cycle regulators such as cyclin D1, cyclin A, p53, p21Cip1, p16Ink4a
and p19ARF. The capacity of AP-1 both to promote and inhibit cell-cycle progression
is most likely due to the abundance of distinct members within a given cell, as well as
the cell type and its microenvironment
Feline OSteosarcoma virus
discovered in Finkel-Biskis-Jinkins (FBJ) Mouse Osteosarcoma
cFos
(con cJun
In AP1 )
vFos che codifica
per 49 aa non presenti
in cFos è più stabile
Nel virus dell’osteosarcoma di topo ,
In topi transgenici dove è espressa in maniera incontrollata
causa disturbi dello sviluppo dello scheletro= ossa lunghe
14q24.3, 380aa, leu-zipper
Aumenta espressione di
DNA 5-methylcytosine
transferase DNMT1 di
4 volte: Quindi, LOF
caderine e molti
soppressori; EMS Bakin, A. V.,
Curran, T. Role of DNA 5-methylcytosine transferase in
cell transformation by fos. Science 283: 387-390, 1999
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MYC P01106: the “sine qua non “ of Burkitt
Myc gene was first discovered in Burkitt's lymphoma
patients. In Burkitt's lymphoma, cancer B cells show
chromosomal translocations, in which Chromosome 8
is frequently involved. Cloning the break-point of the
fusion chromosomes revealed a gene that was similar
to MYeloCytomatosis viral oncogene (110kDa gagvMyc). Thus, the newfound cellular gene was named cMyc.
Myc protein belongs to Myc family of transcription
factors, which also includes N-Myc (neuroblastomas) and
L-Myc genes (small-cell lung cancer). Myc family of
transcription factors contain bHLH/LZ (basic HelixLoop-Helix, Leucine Zipper) domain. Myc protein,
through its bHLH domain can bind to DNA, while the
leucine zipper domain allows the dimerization with its
partner Max, another bHLH transcription factor.
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Esperimento di fusione di Myc a Estrogen Receptor per
dimostrare che la espressione ectopica di myc promuove
proliferazione: potenziale mitogenico di myc
1000 molecole
Emivita 20min
Myc+Max=cell proliferation
2000 geni in
drosophila
Max+MAD=repression, post mitotic differentiation
This experiment illustrates the fact that Myc, acting on
its own and in the absence of serum-associated
mitogens, is able to relieve ALL THE COSTRAINTS on
proliferation that have held these cells in G0 and is able
to shepherd them all the way through G1.
This usually requires substantial, extended stimulation
by growth factors.
Eilers M, Picard D, Yamamoto KR, Bishop JM.
Chimeras of Myc and Steroid Receptors cause hormone
Dependent transformation of cells. Nature 1989; 340: 66-8
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Myc+Max
Myc e Max
Inducono espressione
Di cicline e chinasi
Della crescita G1
Inducono degradazione p27
Myc Reprime (con Miz-1)
espressione di p15INK4
p21Cip, p27Kip
Ras signaling affects MYC via two independent pathways that act synergistically to stabilize
MYC. First, acting through Raf-1 and extracellular signal-regulated kinase (a mitogenactivated protein kinase), Ras phosphorylates MYC at Ser62, thereby extending ...
RICORDATE? RAS
aumenta
La stabilità di Myc
Ser8
SER62
Thr58
Bachireddy P et al. Clin Cancer Res 2005;11:4278-4281
©2005 by American Association for Cancer Research
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Proliferazione di B e traslocazione, Virus Herpes a DNA
Virus tumorale a DNA
cMYC
pS62MYC
Cdk2
pT58MYC
Prolyl
Isomerase
58TPPLSP
pAkt
AKT
Wild type Myc
MYC 439aa è trascritto costitutivamente. Fine tuning è dovuto a
modulazione della stabilità. Myc pT58 viene degradata
Myc regulates glycolysis and glutaminolysis(increase ENERGY)
Increase mTORC1 (increase GROWTH).
Myc activates transcription of all glycolytic enzyme genes
(included the rate-limiting Hexokinase II e Lactate dehydrogenase A)
Myc represses miR-23a, -23b, resulting in greater expression of Mitochondrial Glutaminase
Myc increases Acetyl-CoA, therefore, histone acetylation
Myc increases LEUCINE uptake and alfa keto glutarate (mTORC1 ACTIVATION)
Sloan E J , and Ayer D E Genes & Cancer 2010;1:587-596
Copyright © by SAGE Publications
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Oncogene induced apoptosis/senescence : a feed-back control
1- Myc promotes cell-cycle progression
2- Myc blocks differentiation (Takahashi K, Yamanaka S (August 2006). "Induction of
pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors". Cell 126 (4): 663–76
)
3- Myc promotes Glutaminolysis (Glutamine Addiction)
4- Myc downregulates the angiogenesis repressor
THROMBOSPONDIN
The quartet: cMYC, KLF4, OCT3/4, SOX2 ; NANOG is despensable
5- Ectopic/overexpressed Myc can induce apoptosis !!!???
Mycà ARFà p53
The function of Oct3/4 and Sox2 as core transcription factors to determine pluripotency is well
documented (Boyer et al., 2005, Loh et al., 2006 and Wang et al., 2006). However, they cannot bind
their targets genes in differentiated cells because of other inhibitory mechanisms, including DNA
methylation and histone modifications. We speculate that c-Myc and Klf4 modifies chromatin
structure so that Oct3/4 and Sox2 can bind to their targets (Yamanaka, 2007). Notably, Klf4 interacts
with p300 histone acetyltransferase and regulates gene transcription by modulating histone acetylation
(Evans et al., 2007).
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Glutamine
Addiction
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Myc overexpression in fibroblast provokes
Glutamine addiction
Deficiency in glutamine
and not glucose induces
Myc-dependant apoptosis
in human cells
Published July 2, 2007 // JCB vol. 178 no. 1 93-105
Vari tipi di GAIN OF FUNCTION
Notare :
L’attività di Myc nella trasduzione del segnale dipende
Dalla sua CONCENTRAZIONE nel nucleo.
Le mutazioni che rendono oncogenico Myc ne aumentano I livelli
Oppure allungano l’ emivita (> 25 minuti)
-----------------------------------------------------------------------------------L’attività di Ras, Src, EGFR, PDGFR… dipendono dall’attivazione
Le mutazioni che le rendono oncogeniche colpiscono la loro
REGOLAZIONE
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Conversione in oncogene
Tre meccanismi con cui un proto-oncogene
viene convertito in un oncogene
Double minutes (Mdm2);
HSRs:
homogenously staining
Src, myc region (myc)
myc
Amplification n-Myc in neuroblastoma
Kaplan-Meyer plot of EVENT FREE SURVIVAL
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Human Colon Cancer (COLO-320): Homogenously staining
Chromosomal regions contain amplified copies of an abundantly
Expressed cellular oncogene c-myc in malignant neuroendocrine
cells from a human colon carcinoma
Alitabo, Schwab, Lin, Varmus, Bishop PNAS 1983; 80:1707-11
Homogeneous Staining Region
HSR
BFB
Breakage
Fusione
Bridge
Cycle
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Double minutes DM: small fragments of extrachromosomal DNA
(0,5-10 megabasi)
Mdm2 amplification
Double Minute chromosome: circular, 4 Mb, amplified in Mouse
Fibroblast line 3T3 (1993)
>4 amplification in human leukemiae, osteosarcome and
rhabdomyosarcoma (1995)
Designation:
NCI-H596 [H596] epithelial, human
Constantly, there were 2 to 3 minute chromosomes that were about 1/2 the size of a G group chromosome.
Mdm2 12q13-15 , Ring finger dependent ubiquitin
ligase of p53 and itself
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ARF MDM2 P53
Pokemon : oncogene che reprime specificatamente la trascrizione di
ARF (soppressore, inibitore di MDM2)
Pokemon, which stands for POK erythroid myeloid ontogenic
factor, belongs to the POK family (POZ domain and Kruppel
zinc fingers) of transcriptional factors. If mutated, they become
transcriptional repressors through the engagement of histone
deacetylases, causing chromatin remodeling and in turn
tumorigenesis (2005).
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Receptor overexpression/
overfunction
• Number of genes is amplified
• Elevated transcription (strong promoter)
• Longer half life of receptor protein (e.g., E5 papilloma +EGFR)
• Ectodomain deletion
• Gene fusion causing costitutive dimerization
Receptor dimerization is enhanced
Ligand independent firing is established
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Amplification of erb2/neu oncogene
Breast Carcinoma
Breast carcinoma
EGFR-ErbB1-HER1
1984 EGFR is closely related to oncoprotein erbB discovered
originally in avian ERythroBlastosis virus (AEV)
Ectodomain-less in some
somatic mutations
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Oncogenic EGFR in
Glioblastoma
Poor prognosis in breast, head and
neck, prostate, non-small cell lung
cancer
1
3
2-4 interact to
Form closed
Conformation
1-3 interact to form
Ligand
binding
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)
La overespressione di ErbB2 è tumorigenica perchè
l’eterodimero segnala più efficientemente e più a lungo
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Glioblastoma GBMultiforme
15% tumori intracranici, 60% dei tumori astrocitari/staminali tumorali
Attivazione:
EGFR (ERBB1, HER1, P00533,chr.7)
MDM2
PDGFR
SHP2
Perdita:
TP53
PTEN 50% dei GBM
DCC (Deleted in Colorectal Cancer)
P16
RB
Ring lesion
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[DOI: 10.1126/scisignal.287re6] 2 Science Signaling
Paul H. Huang, Alexander M. Xu and Forest M. White (8 September
Oncogenic EGFR Signaling Networks in Glioma
pPI3K, GAB1 3X
cMET 6X
SHP2 pTyr62 10X
Ras, MEK activated
P27 decreased
pERKdoes not increase
1- Autocrine increase of EGF, TNFalfa
2- Increase of ADAM12 metalloprotease (= more HB-EGF)
3-EGFRvI N-terminal deletion
4- EGFRvII deletion exons 14-15 (Domain 4) open configuration
5- EGFRvIII deleted exons 2-7 (Domain 1,2) costitutive
6- EGFRvIV,vV ligand binding, Y1045 deleted, no CBL
7- Point mutations causing open conformation
-EGFRvIII is constitutively phosphorylated at 10% of the higher extent
-Does not bind CBL because not phosphorylated enough
-When endocytosed, is recycled
-The trasforming ability manfests itself when coexpressed with
oncoRAS, LOH of Ink4A, Arf or PTEN
- EGFRVIII activates PI3K pathway much more than wild type and
independently of EGF
- EGFRVIII increases RAS, MEK (4X), but have low ERK (phosphatase)
- EGFRvIII does not phosphorylate STAT
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Pathways in Invasive Versus Noninvasive GBM
Cells : Tumor heterogenicity
Opposite Activation of Erk
(Proliferation, high in
Core)
and
PI3K/Akt (Invasiveness,
Stemness, higher in Inv)
PH domain Leu-rich repeat Protein
Phosphatases inhibit pAKT
Nestin is
upregulated in Inv,
GFAP in core
Nestin, CD133: markers of
Neural stem cells
GFAP: marker of astrocytic
differentiation
NEURO-Sphere:
Larger in core
(proliferating)
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Recettori
deregolati
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Tropomiosina_Trk Receptor for
basic fibroblast growth factor
Gene fusion= costitutive dimers
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Eredità di oncogeni
Generalmente incompatibile con lo sviluppo embrionale
Fanno eccezione geni ad espressione limitata, localizzata nello
spazio e/o nel tempo ( esempio: RET)
-  Multiple Endocrin Neoplasia 2A e B MEN2A, MEN2B da
mutazioni in RET = REARRANGED DURING
TRANSFECTION
-  PROTOONCOGEN (receptor tyrosine kinase)
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Le traslocazioni
Scambio tra cromosomi non omologhi
1-Geni overespressi
2- Geni nuovi
I tumori spesso originano da
traslocazioni cromosomiche
Linfoma di Burkitt
traslocazione 8:14
Rottura nel
cromosoma 14 in
q32
myc
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Eredità delle traslocazioni
bilanciate
-La eredità di traslocazioni è rara (sono letali all’embrione?)
Quindi si parla di mutazioni SOMATICHE (eccezione:RET,
fibrosarcoma congenito t(12,15) ETV6-NTRK3 (Ets/Tel transcription
factor-NTRK3 ( Neurotrophic Tyr-chinasi)
-Si può ereditare la predisposizione alla traslocazione
(ataxia teleangectasia, ATM)
-337 geni differenti in 358 combinazioni sono noti in
neoplasie umane. Ogni traslocazione è tipica di uno
specifico tumore (a parte t12;15 ETV6-NTRK3)
- Coprono il 20% della “morbidity” (morbosità) dei tumori
(emato, sarcomi, carcinomi)
- Perché, come, quando originano?
- Sono sufficienti a causare tumori?
Oncogenesi da riarrangiamento:
un gene controllato da un promotore forte
Tumore
Linfoma di Burkitt Bcells
c-onc
myc (8)
nuovo promotore
Ig pesante (8 a 14)
Ig leggera (8 a 2/22)
Leucemia linfocitica cronica
a cellule T
myc(8)
recettore cellule T
(8 a22)
Leucemia linfocitica cronica
bcl-2(18)
Ig pesante (18 -14)
a cellule B t(14;18)
bcl-2
Leucemia linfocitica cronica
tcl-1(14)
a cellule T
Ig leggera (18 -2)
recettore cellule T
(inversione 14)
Light Ig lambda in chr. 22; kappa in chr. 2, Heavy chain in chr.14
Bcl=B Cell Lymphoma
TCL= T Cell Leukemia/Lymphoma 1
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La eredità di traslocazioni è rara
(sono letali all’embrione?)
Eccezione : fibrosarcoma congenito t(12,15) ETV6 (Tel, Ets trans
factor-NTRK3 (TYr-chinasi), RET
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Si può ereditare la predisposizione alla traslocazione
(ataxia teleangectasia, ATM)
(Non-Hodgkin lymphoma) In Tumori come CLL Chronic Lymphocytic
Leukemia,
i linfociti sono molti, differenziati= diminuita probabilità di morte
Infatti risulta attivato un antiapoptosi (BCL2 =B CELL LYMPHOMA)
T(14,19) Bcl3-IgH
T(19,22) Bcl3-IgL
T(14,18) Bcl2-IgH
T(2,14) Bcl11A-IgH
In tumori infantili come ALL Acute Lymphocyte Leukemia, i linfociti
sono tanti linfoblasti immaturi= aumentata probabilità di crescita
Infatti risultano attivati fattori di trascrizione o attivatori di
crescita
BCL-2 : B CELL Lymphoma
È un gene che codifica per un fattore antiapoptotico
BCL2 da solo NON è in grado di causare un tumore
Ma
La incapacità ad andare in APOPTOSI, predispone
Le cellule a subire una trasformazione SE un altro
Oncogene viene attivato
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