Il tumore si sviluppa da un’unica cellula
dell’organismo che “impazzisce”, cioè rompe le
regole basilari ed essenziali del
comportamento cellulare fisiologico.
Il singolo clone mutante inizia a proliferare
sino a prendere il sopravvento sulla
popolazione cellulare sana.
Le cellule tumorali hanno due caratteristiche essenziali:
- si riproducono senza rispettare le normali limitazioni della
proliferazione cellulare;
- sono capaci di invadere il tessuto circostante e di formare metastasi
Cancerogenesi
- Il tumore deriva da una singola cellula aberrante;
- Il cancro è causato dal lento accumulo di numerose mutazioni in una
singola linea cellulare (incubazione).
Progressione tumorale:
un lieve disordine iniziale del comportamento cellulare evolve
gradualmente in un cancro conclamato.
Perché sono necessarie numerose mutazioni?
Una cellula per poter diventare tumorale deve acquisire un certo livello
di instabilità genetica, cioè deve mostrare un’aumentata tendenza ad
accumulare mutazioni
Le mutazioni che vanno via via accumulandosi in una cellula
tumorale interessano principalmente quei geni coinvolti
nella regolazione del ciclo cellulare e dell’apoptosi.
Il ciclo cellulare è la serie di eventi che
avvengono in una cellula eucariotica tra una
divisione cellulare e quella successiva.
PROTOONCOGENI e ONCOSOPPRESSORI
PROTOONCOGENI
ONCOSOPPRESSORI
Funzione
•Progressione ciclo cellulare
•Proliferazione cellulare
•Blocco ciclo cellulare
•Apoptosi
•Riparo del danno al DNA
Attività
Proneoplastica
Antitumorale
Mutazione
Dominante
Recessiva
Meccanismo di attivazione
Acquisto di funzione
Perdita di funzione
Alterazione
•Amplificazione genica
•Alterazione regolazione
trascrizionale
•Delezione
•Mutazioni inattivanti
•Ipermetilazione
In condizioni normali la regolazione del ciclo cellulare e della
proliferazione dipende dall’equilibrio tra i prodotti dei protooncogeni e
degli oncosoppressori
PROTOONCOGENI
ONCOSOPPRESSORI
ONCOGENI
cellula normale
ONCOSOPPRESSORI
Mutazioni
Delezioni
Amplificazione
genica
cellula tumorale
L’apoptosi
Aspetti morfologici:
1.
La cellula apoptotica perde i contatti
con le cellule adiacenti
2. Il nucleo si condensa e poi si frammenta
3. La cellula si frammenta in corpuscoli
chiamati corpi apoptotici
4. I corpi apoptotici vengono riconosciuti
e fagocitati dai macrofagi
IAP Family
 c-IAP1
 c-IAP2,
 XIAP
 NAIP
 Apollon
 ML-IAP/livin
 ILP-2
 survivina
Proteine IAP
•
•
•
Numerose copie di domini BIR
RING finger motif al COOH-terminale
Sito di ubiquitinazione
Survivina
•
•
•
•
•
Unico dominio BIR
Ring finger motif assente
α-elica al COOH-terminale
Struttura dimerica
Espressione regolata dal ciclo cellulare
La survivina è presente in tre isoforme diverse derivanti da splicing alternativo:
 survivina full-lenght (142 aa)
localizzazione citoplasmatica
 survivina-2B (165 aa)
 survivina-ΔEx-3 (137 aa)
localizzazione nucleare
Survivin-Δx3 and Survivin-2B: Two Novel Splice Variants of the
Apoptosis Inhibitor Survivin with Different Antiapoptotic Properties
Csaba Mahotka, Michael Wenzel, Erik Springer, Helmut E. Gabbert, and Claus D. Gerharz (1999, Cancer Research)
500 bp
431 bp
329 bp
Differential antiapoptotic potential of the novel survivin splice variants.
Coding sequences of survivin and its two splice variants were ligated into the
expression vector pEGFP-N3 and transfected into the human hepatoma cell
line HepG2. Cell death was induced in transient transfectants by exposure to
methotrexate (100 mg/ml). Survivin-DEx3 transfectants exhibited cell survival
frequencies closely corresponding to that of survivin transfectants. In contrast,
survivin-2B transfectants showed a marked reduction of cell survival.
Electrophoresis of quantitative
RT-PCR products.
(RCC cell line)
L’espressione della survivina è legata al ciclo cellulare
Control of apoptosis and mitotic spindle checkpoint by survivin
Fengzhi Li, Grazia Ambrosini, Emily Y. Chu,Janet Plescia, Simona Tognin, Pier Carlo Marchisio & Dario C. Altieri (1998, Nature)
Cell-cycle synchronization. HeLa cells (ATCC) were treated with 400mM mimosine (to arrest cells in G1), 2mM thymidine (to
arrest cells in S) or 0.4 mgml-1 nocodazole (to arrest cells in G2/M) (Sigma), for 16 h at 37 °C , or were arrested at the G1/S
boundary by sequential culture with 2mM thymidine and 400mM mimosine.
Figure 1 Cell-cycle-dependent expression of survivin in
G2/M. a, Northern blot of survivin (left) or GAPDH (right)
RNA in exponentially growing (no treatment) or drugsynchronized HeLa cells (mimosine, G1; thymidine, S;
nocodazole, G2/M).
Survivin promoter activity. Survivin±luciferase promoter constructs pLuc-230 (-39 to -268), pLuc-cyc1B (-36 to -268) and
pLuc-cyc1.2 (+1 to -268)(numbering from the initiating methionine) were generated by PCR and confirmed by DNA
sequencing. HeLa cells were transiently transfected by LipofectAMINE, synchronized and analysed for luciferase activity in
a Lumat LB 9510 luminometer. Values were normalized for b-galactosidase activity at A405.
Ruolo nella mitosi
Human Survivin Is a Kinetochore-associated Passenger Protein
Dimitrios A. Skoufias, Cristiana Mollinari, Françoise B. Lacroix, and Robert L. Margolis (2000, JCB)
Immunofluorescence microscopy of transfected cells stained with anti–HA (green) and propidium
iodide (red) for chromatin. In different interphase cells, survivin localizes predominantly either in the
cytoplasm (a) or the nucleus (b). In prophase through metaphase (c–e), survivin localizes in distinct spots on
the chromatin. In anaphase, leaves the chromatin, associates with the spindle midzone (f), extending to the
cortex (g), and remains localized in telophase (h) and the midbody (i).
Ruolo nella mitosi
Se la survivina viene silenziata?
• centrosomi sovrannumerari
• fusi multipolari
• interruzione citochinesi
Cellule poliploidi e
multinucleate
Esperimenti su topi knockout
100% morte embrioni
Cell division and cell survival in the absence of survivin
Dun Yang*, Alana Welm, and J. Michael Bishop (2004, PNAS)
Fig. 2. Depletion of survivin elicits nuclear
abnormalities. (A) The effect of survivin RNAi on
cell-cycle distribution. RPE cells were either mocktreated or treated with shRNAs (suv1 and suv1m).
Cell-cycle distribution was analyzed by flow
cytometry 3 days after shRNA transfection. (B)
Nuclear abnormalities in cells treated with survivin
RNAi. Immunostaining of cells was performed 3
days after transfection with suv1 (a–e) or suv1m (
f). Tubulin was detected with fluorescein
isothiocyanate-conjugated mouse anti--tubulin
antibody (green), and nuclei were stained with
DAPI (red). White arrowheads and arrows identify
mininuclei and DNA bridges, respectively. (C)
Percentage of cells with nuclear abnormalities
illustrated in B. Each column represents the
average of three independent experiments, and
each experiment was done in triplicate. More than
500 cells were counted in each measurement.
Error bars represent SD.
metaphase
anaphase
early telophase
late telophase
Fig. 3. Chromosome behavior and
microtubule assembly during mitosis
are abnormal in survivin-depleted
cells.
RPE cells were fixed 2 days after
treatment with either suv1m or suv1.
Immunofluorescence was used to
detect tubulin and survivin (green and
blue, respectively, in the merged
images). DNA was stained with DAPI
(red in the merged images). Cells in
metaphase (a, e, a, and e), anaphase
(b, f, b, and f), early telophase (c, g, c,
and g), and late telophase (d, h, d, and
h) are shown. Arrowheads point to
misaligned chromosomes, red arrows
denote lagging chromosomes or
chromatin bridges, and white arrows
identify visible cortex contraction.
Ruolo nell’apoptosi
Ipotesi sul meccanismo mediante il quale la survivina inibisce
l’apoptosi:
 inibizione diretta della caspasi-3
 interazione con la caspasi-9
 inibizione del complesso pro-apoptotico Smac/DIABLO
Control of apoptosis and mitotic spindle checkpoint by survivin
Fengzhi Li, Grazia Ambrosini, Emily Y. Chu, Janet Plescia, Simona Tognin, Pier Carlo Marchisio & Dario C. Altieri (1998, Nature)
C84A: survivin dominant negative mutant (BIR mutated)
Taxol: microtubule-stabilizing agent
Taxol-induced apoptosis in NIH3T3 transfectants is
inhibited by wild-type survivin, Bcl-2 and XIAP, but not by
the survivin coil-less mutant M1±G99. pcDNA3 is a
control vector.
Displacement of endogenous survivin from microtubules by
forced expression of the C84A BIR mutant resulted in a
progressive increase in caspase-3 activity in synchronized
HeLa cells.
L’α-elica al COOH-terminale e il dominio BIR sono
essenziali per l’attività anti-apoptotica
Regolazione della survivina
La survivina è attivata mediante fosforilazione della Thr34
ad opera della chinasi ciclina-dipendente cdc2-ciclinaB1.
La survivina e’ uno dei geni antiapoptotici trascrizionalmente repressi da p53.
p53 è un fattore di trascrizione che regola il ciclo cellulare e svolge la funzione di
oncosoppressore. E’ infatti in grado di bloccare la progressione cellulare o di
indurre apoptosi.
Nel 50% delle neoplasie p53 è mutata o deleta.
C’è una connessione tra la perdita di p53 wild-type e
l’over-espressione della survivina nel tumore?
Human survivin is negatively regulated by wild-type p53 and
participates in p53-dependent apoptotic pathway
Asra Mirza, Marnie McGuirk, Tish N Hockenberry, QunWu1, Hena Ashar, Stuart Black, Shu Fen Wen2, Luquan Wang1, Paul
Kirschmeier, W Robert Bishop, Loretta L Nielsen, Cecil B Pickett1 and Suxing Liu (2002, Oncogene)
• La survivina è repressa sia trascrizionalmente che a livello proteico da p53
2774qw1: human ovarian cancer cell line expressing mutant p53
Infected with replication-deficient adenovirus expressing wild-type p53
• L’abbassamento dei livelli di mRNA è dovuto alla repressione dell’attività del
promotore della survivina
The human survivin gene has two copies of a p53-binding sequence in its promoter region. These two putative p53-binding
sequences are located at nucleotide position 77 (p53S1) and 71294 (p53S2) relative to the proximal transcriptional start site (+1).
To explore a possible role of these sequences in the repression of survivin gene expression by p53, a series of 5' deletions of the
survivin promoter sequence (nucleotides 71895 to +366) were prepared using PCR.
The indicated survivin promoter constructs were
transiently transfected into 2774 cells in the presence of
wild-type p53 or empty expression vector alone. The
empty luciferase reporter vector (BASIC) was used as a
control for p53 induced non-specific change in
transcription.
Il sito p53S1 è indispensabile per
la repressione da parte di p53?!
The sequence of the p53S1 in the construct Del-2 was mutated by base
substitutions. Transient transfection experiments indicated that these
mutants remained sensitive to p53-dependent repression of luciferase
activity.
La repressione
dell’espressione della
survivina operata da
p53 non è mediata dal
binding al DNA
?
Espressione della survivina nei tessuti normali
La survivina è espressa ad elevati livelli durante lo sviluppo embrionale
ma il gene che la codifica rimane quiescente nella maggior parte dei
tessuti differenziati.
I tessuti differenziati in cui la survivina è espressa (a bassi livelli) sono:
 il timo
 le cellule staminali CD34+ del midollo osseo
 la mucosa gastrica
Il timo
I timociti double-positive (CD4+/CD8+) mostrano overespressione della survivina
mentre questa è espressa a livelli moderati nei single-positive e completamente
assente nei linfociti T maturi.
Le cellule staminali CD34+ del midollo osseo
CD34 è una glicoproteina di membrana che media l’adesione cellula-cellula. E’ espressa
soprattutto negli stadi iniziali del processo di differenziazione dei tessuti ematopoietici.
La produzione di survivina e’ mantenuta costante durante tutto il ciclo cellulare
Espressione della survivina nel tumore
I tumori nei quali è stata dimostrata l’upregolazione della survivina sono i
seguenti:
 polmone
 seno
 colon
 stomaco
 esofago
 pancreas
 utero
 ovaio
 fegato
L’overespressione della survivina nei tumori correla con:
 un fenotipo più aggressivo e invasivo
 una minore responsività agli agenti chemioterapici
Prognosi più infausta
Il livello di espressione della survivina può essere considerato
un fattore predittivo per la risposta al protocollo terapeutico
La survivina come target terapeutico contro il
cancro
La survivina è ritenuta un buon target terapeutico perché:
1.
2.
3.
4.
è richiesta dalla cellula per la proliferazione cellulare
ha attività anti-apoptotica
è espressa ad elevati livelli nei tessuti neoplastici e non in quelli adulti
differenziati
è coinvolta nell’angiogenesi
 RNA interference (siRNA o shRNA)
 Taglio dell’mRNA della survivina tramite un
ribozima mRNA-specifico
 Espressione di un cDNA antisenso per la
survivina
 Espressione di survivina dominant negative
mutant (T34A)
 Utilizzo di piccole molecole antagoniste
(CDK Inhibitors):
•
flavopiridolo: impedisce la fosforilazione
della Thr34
• purvalanolo A: induce la dissociazione
del complesso survivina-caspasi-9
Meccanismo RNA interference
La RNA interference è un meccanismo mediante il quale alcuni frammenti di
RNA a doppio filamento sono in grado di interferire con l'espressione genica.
Survivin Stable Knockdown by siRNA Inhibits Tumor Cell Growth
and Angiogenesis in Breast and Cervical Cancers
Qing-Xia Li1, Jing Zhao, Jia-Yun Liu2, Lin-Tao Jia, Hong-Yan Huang, Yan-Ming Xu, Yong Zhang., Rui Zhang, Cheng-Ji Wang, Li-Bo Yao, Si-Yi Chen,
An-Gang Yang (2006, Cancer Biology and Therapy)
Figure 1. shRNA-mediated inhibition of survivin expression. (A) survivin mRNA levels were determined by RT-PCR in parental SKBr-3
and HeLa cells. (B) Western blot analysis of survivin protein in parental SKBr-3 and HeLa cells.
SKBr-3: human breast cancer cell line; HeLa: cervical carcinoma cell line
The inhibition of survivin gene
expression by RNAi in both
SKBr-3 and HeLa cells can
promote spontaneous apoptosis,
increase apoptotic rates in
response to several proapoptotic
stimuli or resensitize these
cancer cells to chemical drugs.
Che cos’è un ribozima?
Un ribozima è una molecola di RNA in grado di catalizzare una
reazione chimica.
ribozima
Ribozyme-mediated down-regulation of survivin expression
sensitizes human melanoma cells to topotecan in vitro and in vivo
Marzia Pennati, Mara Binda, Michelandrea De Cesare, Graziella Pratesi, Marco Folini, Lorenzo Citti, Maria Grazia Daidone, Franco
Zunino and Nadia Zaffaroni (2004, Carcinogenesis)
The JR8 human melanoma cell line was stably transfected with the active ribozyme RZsurv or the
catalytically inactive ribozyme mutRZsurv.
In vitro cleavage of the 362-nt synthetic 32P-RNA
substrate by RZsurv into the cleavage products (310
and 52-nt). The reaction products were resolved on
5% polyacrylamide/7 M urea gel and visualized by
autoradiography.
JR8/RZsurv cells showed a significantly increased sensitivity to the topoisomerase-I inhibitor
topotecan compared with JR8/mutRZsurv cells.
Fig. 3. Clonogenic cell survival curves obtained after exposure
of JR8/ RZsurv (filled circle) and JR8/mutRZsurv (filled square)
cells to topotecan for 24 h.
Survivin inhibition enhances the antitumor activity of topotecan in vivo.
cDNA antisenso
cDNA antisenso
Induction of Apoptosis and Inhibition of Cell Proliferation by
survivin Gene Targeting
Grazia Ambrosini, Colette Adida, Giorgio Sirugo and Dario C. Altieri (1998, The Journal of Biological Chemestry)
The survivin gene was identified by hybridization with the EPR-1 cDNA, and its coding sequence
was found to be extensively complementary to that of EPR-1
Pulsed field gel electrophoresis and FISH studies suggested the existence of a single EPR-1/Survivin locus spanning 75–130 kb
on chromosome 17q25. The use of single strand-specific probes further demonstrated that EPR-1 and Survivin originated from two
structurally different messages of 1.3 and 1.9 kb
HeLa cells were stably transfected with an EPR-1 cDNA under the control of
an metallothionein-inducible promoter. In these experiments, ZnSO4 induction
of EPR-1 mRNA suppressed the expression of endogenous survivin, which
resulted in massive apoptosis in growth factor-deprived HeLa cells.
9.6%
18.2%
dominant-negative mutant
Inhibition of melanoma tumor growth in vivo by survivin targeting
Douglas Grossman, Paul J. Kim, Jeffrey S. Schechner, and Dario C. Altieri (2001, PNAS)
Expression of a phosphorylation-defective survivin mutant (Thr34Ala) triggered apoptosis in several
human melanoma cell lines and enhanced cell death induced by the chemotherapeutic drug cisplatin in
vitro.
The melanoma cell lines YUSAC2, LOX, and
YUGEN8 were transfected separately with the
indicated GFP-containing plasmids. At 48 h after
transfection, cell nuclei were scored morphologically
as normal or apoptotic by fluorescence microscopy.
Data are expressed as percent apoptosis based on
counting approximately 100 cells per condition
Thr34Ala expressed
Enhancement of cisplatin-induced apoptosis by
expression of survivin Thr34Ala. YUSAC2yT34A-C4 cells
were cultured for 3 days in the presence or absence of tet
and 3 mM cisplatin.
Conditional expression of survivin Thr34Ala in YUSAC2 melanoma cells prevented tumor formation
upon s.c. injection into CB.17 severe combined immunodeficient-beige mice.
When induced in established melanoma tumors, survivin Thr34Ala inhibited tumor growth by 60–70%.
YUSAC2yT34A-C4 tumors were established in 30 animals maintained on
tet. When tumors became apparent (day 0), tet was maintained in the
drinking water of 10 animals (tet +) and withheld from 20 animals (tet -),
and tumors were monitored for 3 weeks.
Withdrawal of Tet in
established tumors was associated with a significant reduction (70-80%)
in growth rate.
Riepilogando…
Grazie a tutti per
l’attenzione!
Irene Bagaloni